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Jos Buttler named ICC Men's Player of the Month (November)
England's white-ball captain Jos Buttler has won the ICC Men's Player of the Month award for November 2022. The swashbuckling batter took his rivals by storm, thereby helping the Three Lions win the ICC T20 World Cup Down Under. Meanwhile, veteran Pakistan batter Sidra Ameen won the award in the women's category, pipping Ireland's Gaby Lewis and Thailand's Natthakan Chantham. Here's more.
Why does this story matter?
- The International Cricket Council (ICC) has been awarding the trophy since 2021.
- The voting panel for the honor features renowned journalists and former cricketers.
- Public voting also has a 10% of stake in the outcome.
- The Player of the Month trophy is handed to the male and female cricketers who did exceedingly well in international cricket across formats in a particular month.
Buttler wins his maiden POTM award
Buttler was on fire in the month of November, clobbering 207 runs in four innings, including a 49-ball 80* (4s: 9, 6s: 3) in the semi-final against India. He piloted England to a 10-wicket win alongside opener Alex Hales. Buttler also smashed a fifty in the group stage against New Zealand. The wicket-keeper batter amassed 225 runs in the tournament Down Under, averaging 45.00.
Buttler beats Adil Rashid, Shaheen Afridi
Buttler beat fellow countryman Adil Rashid and Pakistan's Shaheen Afridi for the honor. Rashid snapped up 1/16 in the must-win group-stage game against Sri Lanka. The leg-spinner claimed 1/20 in the semi-final against India, followed by 2/22 in the final. Shaheen, who returned from a knee injury, concluded as Pakistan's highest-wicket taker in the tournament. The left-arm quick pocketed 11 wickets at 14.09.
Sidra Ameen stands tall!
Ameen bashed 277 runs across three WODIs against Ireland. The opening batter slammed 176* in the first match, followed by 91* in the second outing. She bagged the Player of the Series award as well. She beat ace Thailand batter Chantham and Ireland all-rounder Lewis. Notably, Ameen is the second successive POTM winner from Pakistan, after bowling all-rounder Nida Dar in October.


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Rihanna and LA Dodgers star Matt Kemp are reportedly getting back together. More details inside.
The pair were allegedly spotted out in LA for dinner last week. A source says they saw RiRi and Kemp at an LA restaurant and put into a private dining area. Could this mean Chris Brown is out of the picture or a way to make him jealous?!
In other news, Matt Kemp is all about honoring the legacy of Dr. Martin Luther King Jr. On January 19, Matt kicked off the official MLK parade as the 2013 Celebrity Grand Marshall. Matt cruised through the city of Los Angeles with one of his biggest fans by his side, Judy Henderson, his mother.
In efforts to pay respect to those who made this opportunity possible for him, Matt opted to wear number 42, the jersey of the late Jackie Robinson. Like a true sportsman and a man of the community, Matt chatted with fans, posed for fan photos, and cheered on the crowd with other Dodger Alumni.


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In searching for the diagnosis for the sickly Knicks, there are plenty of symptoms to be found on the offensive end.
They have averaged just 97.8 points per game in their past five contests, which would have entered play Friday as the worst in the NBA as a season total. Their foul shots are down, notching 14 per game through this streak after getting to the line 23 times per contest in their first 11 games. They are shooting worse, there are questions concerning every starter — even RJ Barrett quietly has taken a step back — and a feel-good Knicks season could feel bad in a hurry, especially in a deep East.
But if there is a face of disappointment for a team that has lost four of six, someone who sums up the offensive struggles, it is a player who no longer is even seeing the court late in games.
Evan Fournier was brought in to play good defense, sink a lot of jump shots and be an improvement over Reggie Bullock, who was steady with the Knicks last year and meshed well with emergent star Julius Randle.
The first look was a beauty, when Fournier poured in 32 points in the opening night thriller over the Celtics. But his shot has disappeared since, and he has disappeared in the fourth quarter in the past four games, including Wednesday's letdown loss to the Magic, in which he was subbed out halfway through the third and never reentered.
"Very inconsistent, obviously," Fournier said at Knicks practice Friday about his own start to the season. "Kind of like what we've been doing as a team, to be honest.
"We've had really good games. We've had bad games. … In [15] games, you've seen my best and worst already."
More of the latter, as his 41.3 percent mark from 3 last year has fallen to 35.1 percent this campaign. Particularly troublesome has been his fit in the starting unit alongside Randle, both long-term pieces for the Knicks contractually. The Kemba Walker-Fournier-Barrett-Randle-Mitchell Robinson unit has been 88 points worse than its opponent.
The ball has not moved, Randle has struggled and Fournier has been an afterthought with few chances and fewer chances that have been capitalized upon.
The Frenchman and the Knicks believe the chemistry will come with more time.
"I think after 15 games last season, no one was saying that Julius and Reggie had great chemistry," coach Tom Thibodeau said before the Knicks (8-7) play host to the Rockets on Saturday.
And speaking of time, Fournier needs to earn more of it.
"The way I look at it, I can't just play like I did with other teams knowing I'd play 32 minutes at least," said the 10th-year pro, who received more minutes with Orlando and Boston last year. "I know where my shots are coming from, I know how things are going to go for me. So I can get into the game knowing that.
"I think the situation right now is I don't know how many minutes I'm going to play, so I have to have the mindset of: If I'm going to play 20 minutes, then just come out the gate with extreme energy. Being very alert. Being ultra-aggressive. And trying to have an impact."
He pointed out that historically, he has been a fourth-quarter player, and the stats back it up. The longer he plays in a game, the better he has been. In minutes 30-39 of his game time, he has shot 45.3 percent from the field, which is only bested by his shooting percentage past 40 minutes (48.4).
But if he cannot be aggressive enough and successful enough early on, those late-game moments don't exist.
The Knicks wanted more from a shooting guard/forward signed to a four-year, $78 million pact and now who is averaging 12.2 points. In 2019-20 with the Magic, that number was 18.5.
He is not the only symptom for a team that has lost four of six. Thibodeau referenced the Knicks' recent struggles getting to the line as officials have swallowed more calls and a need to adapt to the changing game. They have been too sloppy with the ball and have shot worse as the season has progressed.
But a hot Fournier would soothe plenty of the pains.
"We're not worried about that. Evan, he's a shooter. He's a great playmaker. Can put the ball on the floor," said Derrick Rose, who is accustomed to getting accustomed to Thibodeau offenses. "I think we just gotta put the emphasis on just getting plays that Evan can get an open look, so he can get a groove going."


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An archetype can refer to an original pattern where things are from the same behavior are copied or based. Therefore, an archetype may also mean a prototype or a recurring symbol, especially from the realms of art and literature.
The "hero within" is a modern psychological book is drawn from anthropology, literature as well as psychology by its author Carol Pearson. The book defines six major heroic types which include the; the magician, the innocent, the warrior, the wanderer, the altruist and finally the orphan. The author shows how people can apply this powerful guide in order to discover their hidden gift to solve our problems and generally to transform our life with the given important resources that are found in our inner strengths (Tallman 87). The book has helped many people to enrich their lives by showing them on how to tap the power of the archetypes that exist within. The book speaks deeply to the hero in us, and that reverberate in all parts of our lives. The book passes a poignant wisdom with several examples. It can also be a tool that can develop our own innate heroic gifts.
In our society, we believe that every great culture has a great warrior tradition that accompanies some of the warrior myths. The Old Testament tells the stories of a warrior people as well as that of a warrior God. I was raised in a community that has perhaps the most legendary warrior tradition. From birth, the great warriors nurtured and trained all boys in the society to become warriors, and that rigorous training developed men of unconquerable spirit (Tallman, p. 83). My archetype has really helped me to fit my own character for several reasons;
This is the first benefits that help me believe that proper use of aggression assist me to have goals that I strive toward. This helps me to have a clear and definite purpose in life.
The archetype of a mindfulness of the Warrior is based on two-fold. First, I am always alert and awake as well as ever vigilant. This has developed my keen situational awareness, and thus I don't let complacency lull me to sleep; instead, I always watch, observe, study, and plan my daily activities. Secondly, my courage is rooted in the fact that I should never be afraid of the death (Pearson 78). I understand that any minute could be my last and thus every day and decision count in my life.
The practice has also helped me to understands my limits and thus take calculation instead of unnecessary risks. The discipline also frees my fear of pain as I was taught that there are bad and good pains in life, but this does not limit me to withstand physical and psychological pain and achieve my goals.
Today the Warrior archetype is seen to apply for those serving in the armed forces, modern books or even in the movies. Therefore, modern culture does not appreciate or recognize those individuals with Warrior energy in the community. The people living in our society have also dumped the romantic ideal of martial courage (Pearson 75). On the other hand, the archetype has helped me to avoid confrontation and conflict and instead become a nice guy in the society.
The problem associated Warrior energy is that the Warrior energy may not appliy with the other masculine archetypes or even directed by empathy, contemplation, and order. As a result, the society pushes such people to be sensitive because they fear us from becoming abusive and destructively angry.
The Magician is an archetype I desire to acquire as it is associated with alchemy, mystery, and transformation. In the shadow form, the archetype can be used to deceive, distract and manipulate through the act of altering several perceptions of reality. I also desire to have this archetype as the Magician is believed to possess the innate relationship with potentiality and possibility. The Magician is also recognized as a thinker, a weaver, a visionary, an intuitive and a creator of sacred space. A Magician can also be powerful or dynamic archetype with different variants. Individuals with such kind of archetype usually have an interest or specialization in astrology, quantum physics, psychotherapy, mythology, technology, mysticism and other esoteric fields. Magician are known to have an intellect, and thus they can easily connect the inner world of spirit and mind to that of the outer world of creation as well as that of physical manifestation (Pearson 117).
The outer expression of the archetype Magician can be acquired by a constant learner. To achieve this, one can drive or gain this knowledge of understanding the outside world through books, teachers or applying other methods of formal education. The inner expression of the Magician can also be acquired through intuition (Pearson 132). The Magician learns from the inner experience of intuiting and trusts your inner information through action.
Work Cited
Pearson, Carol. Awakening the Heroes Within: Twelve Archetypes to Help Us Find Ourselves and Transform Our World. San Francisco: HarperSanFrancisco, 1991. Internet resource.
Tallman, Bruce. Archetypes for Spiritual Direction: Discovering the Spiritual Heroes Within. New York: Paulist Press, 2005. Print.


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Mindfulness approaches, arising from Eastern practices, have been recognised as a third-wave of psychotherapy in Western societies because of its ability to improve psychological functioning and to reduce emotional issue across a wide range of illness. Mindfulness-based therapies consist of a wide range of techniques suitable to the needs of individuals, similar to Cognitive Behavioural Therapy. This subject explores the core elements of mindfulness a variety of mindfulness techniques and the relationship between mindfulness and de-automatisation.
One session
School of Biomedical Sciences
For any enquiries about subject selection or course structure please contact Student Central or firstname.lastname@example.com or phone on 1800 275 278.
For further information about Charles Sturt University, or this course offering, please contact info.csu on 1800 275 278 (free call within Australia) or enquire online.
The information contained in the 2018 CSU Handbook was accurate at the date of publication: August 2018. The University reserves the right to vary the information at any time without notice.


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Micropulse Laser Treatment Works Across Glaucoma Subtypes
Researchers reviewed the efficacy of micropulse transscleral cyclophotocoagulation on different glaucoma subtypes.
Researchers reviewed the efficacy of micropulse transscleral cyclophotocoagulation on different glaucoma subtypes.
Dr. Ehrlich reviews the evidence of 3 recent studies that evaluate the risk of fracking and health outcomes in infants, management of hypertension in pregnancy, and cardiac risk of eye surgery.
Recognize the signs and symptoms of red eye that differentiate benign from vision-threatening ophthalmologic illnesses.
A man in his late 40s comes to the emergency department with a complaint of blurry vision that lasted for approximately 20 minutes before returning to normal. The loss of focused vision affected both eyes. Can you make the diagnosis?
A 22-year-old man is brought to the emergency department for the management of facial trauma sustained during an assault at a nightclub.
Recent guidelines are designed to optimize uveitis screening protocols for pediatric patients.
EyeArt is the first FDA-cleared AI technology for autonomous detection of more than mild diabetic retinopathy and vision-threatening diabetic retinopathy.
Children should be screened for amblyopia before the age of 5.
Cannabis may disrupt and delay signal transmitted along the visual pathway via the optic nerve.
Greater intake of dietary nitrate and green leafy vegetables was associated with a 20 percent to 30 percent lower risk of primary open-angle glaucoma, according to a study published online by JAMA Ophthalmology.


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The 50TH NAACP Awards Review
The fresh NAACP Image Honors is actually a great fun time and constantly great celebrating what folks of colour give the fresh table. The newest gains began from the low-televised eating stored at Beverly Hilton in Beverly Slopes, California after which continued onto the live show in the Dolby Movies for the Movie industry, California.
Among gains, the most significant went to Black colored Panther, truly and additionally along. Chadwick Boseman, A great Star into the a film. Michael B. Michael jordan, Outstanding Supporting Actor for the a film. Danai Gurira, A fantastic Help Celebrity in within the a film. Ryan Coogler, A good Pointing within the a movie. Ryan Coogler and Joe Robert Cole, A good Writing in a movie. Letitia Wright, A great Breakthrough Overall performance in the a film. The Sound recording/Complilation. A fantastic Dress Shed in the a motion picture. Last but not least, Outstanding Motion picture. Strategy to use party. Instance a great movie you to is definitely worth all of the detection it's got gotten.
Black-ish was also among larger winners towards evening and An excellent Comedy Collection. Energy took household the A good Drama Series. Maxine Waters who acquired the latest Chariman's Prize offered all of us a comprehensive course inside the political issues, while Jay-Z is recognized on Presidents Award and you will Beyonce' took home Performer of the year. It absolutely was an effective nights.
Huge REINVENTED That have 'LITTLE'
Had an opportunity to pick Little during the a young tests. Nothing are an entertaining experience regarding start to finish. If you failed to remember that Regina Hallway are comedy after that rating in a position on the ride. Yes, generally Regina takes on the fresh new siddity uppity method of however, this time around she provides funny to itbine that with the newest comedic timing away from Issa Rae and Marsai Martin and you've got an absolute combination. We foresee that is various other profit to possess movie star manufacturer Usually Packer and you can an earn having Marsai Martin who's the brand new youngest exec manufacturer using this type of movie.
Jordan is a take no-inmates employer exactly who helps make her secretary and you can employees lifestyle a full time income hell each day. She confronts an urgent possibilities whenever a young child desires you to definitely she try nothing. The lady life and you can field turn upside down whenever she amazingly converts with the a beneficial thirteen-year-old sorts of herself.
Absolutely nothing 's the Slutty Tuesday into the Black colored people. Story of the Tracy Oliver and directed by the Tina Gordon, Little requires all of us towards a trip regarding investigating and dealing having thinking and you can thinking you to Michael jordan never dealt with in the past. It entails her returning to the full time whenever she felt powerless and you can believed unwanted. People can be vicious which end up in their hard external and the inability to let anyone set for concern with being harm once more. Immediately following Michael jordan try forced to end up being an excellent thirteen-year-old once more she found that something hadn't altered and you will pupils remained horrible. But, it was whenever she grabbed their strength back and befriended the new infants that have been just like the woman since the a child that she understood she had a create more than and you can showed him or her it was ok becoming other. Nothing was a reminder to deal with all of our ideas and you will thinking in advance of it fester and now have out of control, and be the ideal types of ourselves.
This film has actually a fantastic shed whoever chemistry helps make the film really works. Little starts that have Michael jordan are awakened inside her penthouse flat within the Atlanta from the her current invention for her profitable tech organization, Family Girl that's like Amazon's Alexa and voiced from the not any other than just Black colored-ish star and funny woman Tracee Ellis Ross. In early stages we discover Jordan's like notice starred from the Luke James and are usually handled so you can an attractive dance results away from him. Vocal and you may dancing, yes so you can both. These are Luke James, let us discuss the most other eye candy within this film, Justin Hartley, out of this is United states. I became proper with little Jordan when she said "you feel just what I am effect." Rounding out this new throw are Tone Bell who's Issa Rae's reputation, April greatest supporter and you may JD McCrary exactly who brings a great results at the college or university skill show when you find yourself Michael jordan are nothing. Let me as well as scream the incredible closet agencies, flames throughout the flick.
Tracy plus existed just after to speak with men, offered incredible recommendations and you may sense, and take photographs. The girl future strategies https://datingranking.net/es/mexican-cupid-review/ include an use the film, First Wives Club and you can Unaware. Happy observe exactly what otherwise she's got upwards their sleeve.


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Browsing: up panchayat election
'Why Panchayats who die of Corona on the 31st day of election duty are not entitled to ex-gratia?' Allahabad High Court seeks reply from UP government
The lawyer told the court that no such standard can be set that the corona infected employee will die in…
UP District Panchayat President Election: BJP candidates will be elected unopposed in 17 districts, June 29, the last day for withdrawal of nominations
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Now the village head will be able to easily call a meeting in UP, 'Apna Panchayat Bhawan' is going to meet all the panchayats of the state
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Cases registered in 301 violent incidents, 10 murders occurred during UP Panchayat Election, police revealed
According to the police, a total of 485 criminal cases were registered in the last 2015 panchayat elections. Of these,…
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UP Panchayat Election: 3,27,036 candidates declared elected in Uttar Pradesh Panchayat elections
According to the commission, counting of votes is going on peacefully in 826 centers in 75 districts of Uttar Pradesh.…


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Desta: The Memories Between
Desta: The Memories Between is a turn-based strategy video game developed and published by ustwo games.
Desta: The Memories Between is a character-driven rogue-like, featuring an immersive story, strategic turn-based tactics and… a metaphorical ball-game?
From the creators of Monument Valley, Desta: The Memories Between is a story of broken relationships, words left unsaid and the chance to explore your own dreams for answers.
Key Features
- Explore Unfolding Dreams – Every night, Desta falls into the mysterious dreamworld, filled with shattered remnants of locations past, memories of regret-filled relationships, and powerful orbs that can change the course of conversation.
- Play a Surreal Ball Game – Overcome tactical challenges with perfect throws, shoot spirited trick shots and land sensational hits in turn-based sports-style encounters. As nights progress, Desta will become more experienced and attuned to this surreal world, discovering new abilities and ways to win.
- Meet Powerful Allies – Brought to life with a full voice cast, Desta will meet all sorts of interesting and strange characters throughout their journey, all with exceptional abilities and powers, each with their own story to tell about their connection to this world
- Introduction to Tactics – From beginning to end, being approachable is a core value of all ustwo games. Desta: The Memories Between is designed for players of all skill levels to be able to experience the full journey
This game contains themes of self-reflection, anxiety, mental health, loss of parental figures and gender identity. Player discretion is advised.
Desta: The Memories Between News
Desta: The Memories Between for Switch, PC launches in early 2023
Roguelike game Desta: The Memories Between, which first launched for iOS via App Store and …
ustwo games announces turn-based strategy game Desta: The Memories Between
Monument Valley developer ustwo games has announced Desta: The Memories Between, a turn-based strategy game …
Desta: The Memories Between GalleriesView full gallery
June 9, 2022


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research papers
Structural and functional investigation of the human snRNP assembly factor AAR2 in complex with the RNase H-like domain of PRPF8
aLaboratory of RNA Biochemistry, Freie Universität Berlin, Takustrasse 6, 14195 Berlin, Germany, bLaboratory of Structural Biochemistry, Freie Universität Berlin, Takustrasse 6, 14195 Berlin, Germany, and cMacromolecular Crystallography, Helmholtz-Zentrum Berlin für Materialien und Energie, Albert-Einstein-Strasse 15, 12489 Berlin, Germany
*Correspondence e-mail: email@example.com, email@example.com
Small nuclear ribonucleoprotein complexes (snRNPs) represent the main subunits of the spliceosome. While the assembly of the snRNP core particles has been well characterized, comparably little is known of the incorporation of snRNP-specific proteins and the mechanisms of snRNP recycling. U5 snRNP assembly in yeast requires binding of the the Aar2 protein to Prp8p as a placeholder to preclude premature assembly of the SNRNP200 helicase, but the role of the human AAR2 homolog has not yet been investigated in detail. Here, a crystal structure of human AAR2 in complex with the RNase H-like domain of the U5-specific PRPF8 (PRP8F RH) is reported, revealing a significantly different interaction between the two proteins compared with that in yeast. Based on the structure of the AAR2–PRPF8 RH complex, the importance of the interacting regions and residues was probed and AAR2 variants were designed that failed to stably bind PRPF8 in vitro. Protein-interaction studies of AAR2 with U5 proteins using size-exclusion chromatography reveal similarities and marked differences in the interaction patterns compared with yeast Aar2p and imply phosphorylation-dependent regulation of AAR2 reminiscent of that in yeast. It is found that in vitro AAR2 seems to lock PRPF8 RH in a conformation that is only compatible with the first transesterification step of the splicing reaction and blocks a conformational switch to the step 2-like, Mg2+-coordinated conformation that is likely during U5 snRNP biogenesis. These findings extend the picture of AAR2 PRP8 interaction from yeast to humans and indicate a function for AAR2 in the spliceosomal assembly process beyond its role as an SNRNP200 placeholder in yeast.
Keywords: spliceosomal assembly; U5 snRNP; AAR2; PRPF8.
1. Introduction
Precursor messenger RNA (pre-mRNA) splicing is catalyzed by a highly dynamic, multi-megadalton ribonucleoprotein (RNP) machinery, the spliceosome (Will & Lührmann, 2011; Wahl et al., 2009). The small nuclear RNPs (snRNPs) U1, U2, U4, U5 and U6 are the main subunits of the major, U2-type spliceosome. Each of these snRNPs contains a particle-specific snRNA, seven common Sm proteins [or like Sm (LSm) proteins in the case of U6] and a set of particle-specific proteins (Will & Lührmann, 2001). The U5 snRNP is the only snRNP subunit that is also employed by the minor spliceosome (Will & Lührmann, 2005). Apart from the snRNPs, a multitude of proteins and protein complexes that are not stably associated with an snRNP also join the spliceosome to facilitate and regulate pre-mRNA splicing (Agafonov et al., 2011). For each round of splicing, the spliceosome assembles anew on a pre-mRNA by the stepwise association of snRNPs and non-snRNP proteins. Its catalytic center is not preformed but only emerges during assembly by repeated, extensive remodeling of the specific RNA–protein interaction network of each assembly stage to eventually elicit intron excision and exon ligation via two transesterification reactions, referred to as step 1 and step 2 (Wahl et al., 2009; Will & Lührmann, 2011).
SnRNPs themselves are assembled via complex pathways, which in the cases of U1, U2, U4 and U5 include cytoplasmic and nuclear phases (Will & Lührmann, 2001; Matera & Wang, 2014; Gruss et al., 2017). The corresponding snRNAs are synthesized by RNA polymerase II (Pol II), modified with an m7G cap, processed by the integrator complex and exported to the cytoplasm. Here, the Sm proteins are assembled stepwise to form a ring-like structure around a U-rich Sm site in the snRNAs via the protein arginine methyltransferase 5 complex and the survival motor neuron (SMN) complex. Trimethylguanosine synthase 1 then catalyzes hypermethylation of the m7G cap to generate an m2,2,7G cap. The hypermethylated cap and the assembled Sm core domain act as a composite nuclear localization signal that facilitates re-entry of the Sm core RNPs into the nucleus.
The integration of particle-specific proteins into snRNPs also requires specific assembly factors and chaperones. For example, in human cells, the adaptor protein, nuclear FMR1-interacting protein 1, and the heat-shock protein 90 (HSP90)/Rvb1–Rvb2–Tah1–Pih1 (RT2P) chaperone machinery in collaboration with a nuclear-localized SMN complex facilitate integration of the U4/U6-specific proteins NHP2-like protein 1 and pre-mRNA processing factor (PRPF) 31 into the U4/U6 di-snRNP (Bizarro et al., 2015). The HSP90/RT2P chaperone machinery also supports the assembly of a U5 snRNP module composed of the PRPF8 protein (Prp8p in yeast), the 116 kDa U5 small nuclear ribonucleoprotein component (EFTUD2; Snu114p in yeast), the U5 small nuclear ribonucleoprotein 200 kDa helicase (SNRNP200; Brr2p in yeast) and the SNRNP40 protein in the cytoplasm, thereby promoting formation of the mature U5 snRNP (Malinová et al., 2017). Several additional proteins have been implicated in U5 snRNP or U4/U6-U5 tri-snRNP assembly in metazoans (Malinová et al., 2017; Klimešová et al., 2021; Erkelenz et al., 2021; Bergfort et al., 2022).
Some snRNPs are profoundly remodeled during pre-mRNA splicing, necessitating specific recycling mechanisms to reassemble the particles for further rounds of splicing. For example, during spliceosome activation the U4 and U6 snRNAs, which are extensively base-paired in the U4/U6 di-snRNP, are unwound by the SNRNP200 helicase, and U4 snRNA and all U4/U6-associated proteins are displaced (Laggerbauer et al., 1998; Raghunathan & Guthrie, 1998; Agafonov et al., 2011). Furthermore, in human cells the U5 snRNP enters the spliceosome as a 20S particle but is released as a 35S particle after splicing due to incorporation of the PRPF19 complex and additional factors (Makarov et al., 2002). Late de novo snRNP biogenesis steps and recycling of snRNPs after pre-mRNA splicing take place in nuclear Cajal bodies (Staněk & Neugebauer, 2004, 2006; Sleeman et al., 2001; Sleeman & Lamond, 1999).
The U5-specific PRPF8 protein is one of the most conserved nuclear proteins and coordinates proteins, snRNAs and the pre-mRNA at the catalytic center of the spliceosome (Grainger & Beggs, 2005). PRPF8 harbors two regulatory pseudo-enzyme domains at its very C-terminus, comprising an RNase H-like (RH) and a Jab1/MPN-like (JM) fold (Pena et al., 2007, 2008; Zhang et al., 2007; Yang et al., 2008; Ritchie et al., 2008). In yeast, the A1 cistron-splicing factor (Aar2p) has been characterized as a U5 snRNP assembly and recycling factor that mediates the formation of pre-U5 snRNPs lacking the Brr2p helicase in the cytoplasm (Gottschalk et al., 2001; Boon et al., 2007). Aar2p concomitantly binds the RH and JM domains of Prp8p (Weber et al., 2011, 2013; Galej et al., 2013). By sequestering the Prp8p JM domain, which is a major binding site of the Brr2p RNA helicase, Aar2p initially prevents the integration of Brr2p into U5 snRNP (Weber et al., 2013; Galej et al., 2013). Aar2p accompanies the Brr2p-deficient pre-U5 snRNP particle into the nucleus, where phosphorylation of its Ser253 residue triggers refolding and release of Aar2p, allowing Brr2p entry to complete U5 snRNP biogenesis (Boon et al., 2007; Weber et al., 2013).
In a previous study, we demonstrated that the human Aar2p homolog AAR2 is produced from the c20orf4 gene in HeLa cells and that it stably binds the PRPF8 RH domain in vitro (Santos et al., 2015). However, human AAR2 and yeast Aar2p exhibit only 24% sequence identity, questioning the extent to which their structures and molecular mechanisms are conserved. While proteomics studies and pull-down experiments have suggested that human AAR2 also participates in U5 snRNP assembly (Malinová et al., 2017; Klimešová et al., 2021), human AAR2 has been identified in a complex with PRPF8, EFTUD2, SNRNP200 and SNRNP40 (Malinová et al., 2017), indeed indicating potential differences in the Aar2p/AAR2-mediated U5 snRNP assembly steps in yeast and humans.
Here, we report a co-crystal structure of human AAR2 in complex with the PRPF8 RH domain (PRPF8RH) and present further interaction studies of AAR2 with PRPF8 fragments and the SNRNP200 helicase in vitro. In contrast to the situation in yeast, we find that a human AAR2–PRPF8RH complex does not bind the PRPF8 JM domain and thus permits the formation of a trimeric AAR2–PRPF8–SNRNP200 complex. As in yeast, the human AAR2–PRPF8RH interaction is abrogated in vitro by a phosphomimetic S284E (S253E in yeast) mutation, indicating highly conserved regulation of AAR2 by phosphorylation. Furthermore, AAR2 seems to lock PRPF8RH in its first-step conformation and block the conformational switch to a step 2-like, Mg2+-coordinated conformation during U5 snRNP biogenesis. Our results shed the first light on the human AAR2–PRPF8RH interface and imply a different role of AAR2 in spliceosomal assembly than in yeast.
2. Materials and methods
2.1. Cloning, expression, protein purification and reconstitution of protein complexes
We employed a modified pFL vector encoding a truncated version of human SNRNP200 lacking the first 394 residues (residues 395–2136) and containing an N-terminal, TEV-cleavable His10 tag (Santos et al., 2012), a pET-M11 plasmid encoding human PRPF8RH (residues 1747–2016) and containing an N-terminal, TEV-cleavable His6 tag (Pena et al., 2008; Weber et al., 2011), a pET-M11 plasmid encoding human PRPF8JM (residues 2064–2335) and containing an N-terminal TEV-cleavable GST tag (Mozaffari-Jovin et al., 2013), and pFL vectors encoding human AAR2 or AAR2Δloop (AAR2 with residues 170–200 replaced by three serines) with both AAR2 constructs containing an N-terminal, TEV-cleavable His10 tag (Santos et al., 2015), which have been described previously. The inserts were derived from codon-optimized synthetic genes. A DNA fragment encoding the C-terminal fragment of PRPF8 (encompassing the RH and JM domains; PRPF8RH-JM; residues 1760–2335) was PCR-amplified from a codon-optimized prpf8 synthetic gene and cloned into the pIDK donor vector by restriction-enzyme cloning using XhoI and NdeI (NEB). Snrnp200395–2136-pFL and prpf81760–2335-pIDK were fused by Cre-Lox recombination and used for bacmid preparation. A codon-optimized DNA fragment encoding human AAR2 residues 1–364, lacking the C-terminal 20 residues of AAR2, was cloned into a modified pFL vector by restriction-enzyme cloning using EcoRI and HindIII (NEB) to guide the production of AAR21–364 containing a C-terminal His6 tag. A codon-optimized DNA fragment encoding human AAR2 was cloned into pcDNA3.1(+) using BamHI and XhoI restriction enzymes (NEB) to guide the production of AAR2 containing an N-terminal FLAG tag. Mutations were introduced with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. All constructs were verified by dye-terminator sequencing (Seqlab).
Purified human SNRNP200395–2136 (8 mg ml−1; Santos et al., 2012), PRPF8RH (30 mg ml−1; Pena et al., 2008), PRPF8JM (10 mg ml−1; Mozaffari-Jovin et al., 2013), AAR2 (12 mg ml−1; Santos et al., 2015), AAR2Δloop (12 mg ml−1; Santos et al., 2015), an SNRNP200395–2136–PRPF8JM complex (Mozaffari-Jovin et al., 2013; 8 mg ml−1) and an AAR2–PRPF8RH complex (20 mg ml−1; Santos et al., 2015) were obtained as described previously. AAR21–364 (11 mg ml−1) was produced and purified as described for AAR2 but with omission of the TEV protease cleavage step, leaving the His6 tag intact. SNRNP200395–2136 and PRPF8RH-JM were co-produced based on a recombinant baculovirus derived from the recombined bacmid in Sf9 cells. For purification of the SNRNP200395–2136–PRPF8RH-JM complex, cells were resuspended in resuspension buffer [50 mM Tris–HCl pH 8.0, 300 mM NaCl, 5%(v/v) glycerol, 1 mM DTT, 0.05%(v/v) NP40] supplemented with EDTA-free protease inhibitors (Roche) and DNase I (NEB) and lysed via sonification. After centrifugation, the lysate of about 50 column volumes was filtered and passed through Ni–NTA beads (Qiagen). The beads were washed twice with ten column volumes of resuspension buffer containing 15 mM imidazole. The captured complex was eluted with two column volumes of resuspension buffer containing 500 mM imidazole, TEV protease (0.5 mg per millitre of protein solution) was added and the mixture was dialyzed against 20 mM HEPES–NaOH pH 7.5, 200 mM NaCl, 1 mM DTT overnight at 4°C. Five column volumes of the buffer-exchanged sample were again passed through Ni–NTA beads and the flowthrough was collected. The complex was concentrated to a final concentration of 7 mg ml−1 and further purified by size-exclusion chromatography (SEC) on a Superdex S200 10/300 column (GE Healthcare) in 20 mM HEPES–NaOH pH 7.5, 200 mM NaCl, 1 mM DTT.
2.2. Analytical size-exclusion chromatography
Individual proteins and protein mixtures were analyzed by analytical SEC on a Superdex 200 Increase PC3.2/30 column (GE Healthcare) in 20 mM Tris–HCl pH 7.5, 250 mM NaCl, 0.5 mM DTT at a flow rate of 50–70 µl min−1. For analysis of complex formation, proteins (at the concentrations stated in Section 2.1) were mixed in equimolar ratios in 60 µl size-exclusion buffer and incubated for 30 min on ice. Elution fractions were supplemented with SDS–PAGE loading buffer and analyzed by SDS–PAGE.
2.3. Crystallographic analyses
Crystallization of the human AAR2Δloop–PRPF8RH complex has been described previously (Santos et al., 2015). Briefly, 1 µl purified human AAR2Δloop–PRPF8RH complex concentrated to 14 mg ml−1 in 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5 mM DTT was crystallized employing an equal volume of reservoir solution consisting of 0.1 M HEPES pH 7.0, 10%(w/v) PEG 6000, 5%(v/v) 2-methyl-2,4-pentanediol. Crystals were transferred to reservoir solution supplemented with 10%(v/v) 2-methyl-2,4-pentanediol and flash-cooled in liquid nitrogen. Diffraction data were collected on beamlines BL14.1, BL14.2 and BL14.3 of the BESSY II storage ring, Berlin, Germany and on beamline P14 of the PETRA III storage ring, Hamburg, Germany at 100 K and were processed with XDS (Kabsch, 2010). The structure was solved by molecular replacement with Phaser (McCoy et al., 2007) using chain A of the structural coordinates of human PRPF8RH (PDB entry 3e9l; Pena et al., 2008), omitting the water molecules. An initial model of the AAR2Δloop subunit was obtained by automated model building with phenix.autobuild (Adams et al., 2010). The model was completed through alternating rounds of automated refinement using phenix.refine (Afonine et al., 2012) and manual model building using Coot (Emsley et al., 2010). The quality of the final model was assessed with MolProbity (Chen et al., 2010). Of note, a relatively large number of difference density peaks were observed in the Fo − Fc map. Structural figures were prepared with PyMOL (Schrödinger).
3. Results
3.1. Crystal structure of a human AAR2Δloop–PRPF8RH complex
Structures of yeast Aar2p in complex with the Prp8p RH and JM domains or with full-length Prp8p have been reported (Weber et al., 2011, 2013; Galej et al., 2013). In contrast, the structure of human AAR2, which exhibits only 24% sequence identity to the yeast ortholog (Supplementary Fig. S1), and the structural basis for its interaction with PRPF8 remain unknown. We previously reported the crystallization of a human AAR2 variant in which an internal loop (residues 170–200) was replaced by three serine residues (AAR2Δloop) in complex with PRPF8RH (Santos et al., 2015). In yeast Aar2p, the corresponding internal loop was shown to hinder crystallization, to be protease-cleavable and to be irrelevant for the interaction with Prp8p C-terminal domains (Weber et al., 2011, 2013; Santos et al., 2015). Here, we report the crystal structure of the human AAR2Δloop–PRPF8RH complex at 2.35 Å resolution. The structure was solved by molecular replacement with Phaser using the structural coordinates of PRPF8RH (PDB entry 3e9l; Pena et al., 2008) as a molecular-replacement search model (Table 1).
‡CC1/2 = (〈I2〉 − 〈I〉2)/(〈I2〉 − 〈I〉2) + , where is the mean error within a half data set (Karplus & Diederichs, 2012).
§Rwork = (for the working set; no σ cutoff was applied).
¶Rfree is the same as Rwork but calculated on the test set of reflections that were excluded from refinement.
††Estimated overall coordinate error based on maximum likelihood.
‡‡Calculated with phenix.refine (Afonine et al., 2012).
§§Root-mean-square deviation.
Apart from ten N-terminal residues, the region spanning residues 159–201 including the three serines connecting residues 170–200 of the internal loop, a loop connecting helices α9 and α10 (residues 313–321) and six C-terminal residues of AAR2Δloop (Supplementary Fig. S1), all residues of AAR2Δloop and PRPF8RH could be reliably modeled into well defined electron density (Supplementary Fig. S2). Residues 65–71 of AAR2 and residues 2001–2008 of PRPF8RH were modeled with low confidence due to weaker electron density.
Despite the low sequence identity, the overall structure of human AAR2Δloop in the AAR2Δloop–PRPF8RH complex is very similar to that of yeast Aar2pΔloop in complex with the Prp8p RH and JM domains (Weber et al., 2013; Galej et al., 2013; root-mean-square deviation of 2.13 Å for 236 pairs from 330 AAR2 and 342 Aar2p Cα atoms; Supplementary Fig. S3). As previously observed for Aar2p (Weber et al., 2011, 2013; Galej et al., 2013), human AAR2Δloop exhibits an N-terminal domain (NTD; residues 10–158) mainly composed of β-strands, an α-helical C-terminal domain (CTD; residues 202–364) and a C-terminal, irregularly structured tail (residues 365–384) (Fig. 1a).
In the full-length yeast Aar2p–Prp8p structure (PDB entry 4i43; Galej et al., 2013), the C-terminal peptide of Aar2p is fully structured and contacts several other Prp8p domains. Superposition with the present human AAR2–RH complex structure suggests that due to the shorter AAR2 C-terminal peptide in humans, contacts with other PRPF8 domains may be limited. Hence, the differences in the functionally important C-terminal peptide of yeast and human Aar2p/AAR2 may hint at a somewhat different mode of action of AAR2 in U5 snRNP or U4/U6-U5 tri-snRNP assembly in humans.
Despite the overall structural similarity of both individual components, the protein interfaces between yeast and human Prp8pRH/PRPF8RH and Aar2p/AAR2 are markedly different. As in yeast, the NTD lacks direct interactions with PRPF8RH, while the CTD and the C-terminal tail of AAR2 establish two interfaces with PRPF8RH (interfaces I and II, respectively; Figs. 1a–1c). In interface I, an edge of the AAR2Δloop CTD laterally contacts PRPF8RH (Figs. 1d and 1e). Interface II is built by the C-terminal residues 366–377 of AAR2 extending across the PRPF8 RH domain below the protruding β-finger module (Figs. 1f and 1g). Both interfaces bury a comparable surface area in the yeast and human systems (interface I, 399 and 412 Å2, respectively; interface II, 733 and 511 Å2, respectively).
Interface I is dominated by hydrophobic contacts, with only four of 12 PRPF8RH-interacting residues conserved between yeast Aar2p and human AAR2, underlining the different organization of the interactions. The conserved core of interface I includes interactions between Ile225 of AAR2 (Ile189 in Aar2p) and Val1874 of PRPF8RH (Val1946 in Aar2p) as well as between Met230 of AAR2 (Met195 in Aar2p) and Trp1839 of PRPF8RH (Trp1911 in Aar2p) (Figs. 1d and 1e; Supplementary Fig. S1). Compared with yeast Aar2p, the AAR2 CTD harbors two extended helices (α11 and α12; Supplementary Fig. S3). In addition, Ile225 and Met230 are shifted by four residues (about one helical turn) along the α5 helix compared with the equivalent residues in yeast Aar2p (Figs. 1d and 1e), giving rise to a markedly different angle with which human AAR2Δloop contacts the PRPF8 RH domain compared with yeast Aar2pΔloop in the Aar2pΔloop–Prp8pRH–Prp8pJM complex (Fig. 1c). Also, the AAR2Δloop residues participating in interface II are only partially conserved between yeast and humans (two of eight residues; Supplementary Fig. S1).
3.2. Similarities and differences in AAR2–PRPF8–SNRNP200 and Aar2p–Prp8p–Brr2p interactions in humans and yeast
The low degree of conservation of AAR2 and observed marked differences in the interface with PRPF8RH have apparent consequences for AAR2 function and likely for interactions within the spliceosome. To test the importance of the specific contacts between AAR2Δloop and PRPF8RH that are observed in our co-crystal structure, we conducted analytical SEC runs with wild-type (WT) proteins and variants. To this end, we investigated the binding of WT AAR2 to WT PRPF8RH in previous work, which is only shown here for comparison (Figs. 2a–2c; Santos et al., 2015). In yeast, the C-terminal tail of Aar2p is dispensable for Prp8RH binding (Weber et al., 2011). Conversely, in the human system, AAR21–364, which lacks the C-terminal tail, no longer binds stably to PRPF8RH (Figs. 2a–2d). Likewise, converting Trp1839 of PRPF8RH or Met230 of AAR2, which are part of the conserved core of interface I, individually to alanine residues abrogated complex formation (Fig. 2e and 2f). Again, the situation differs in yeast, where only Trp1911 of Prp8pRH (equivalent to Trp1839 in human PRPF8RH), but not Met195 of Aar2p (equivalent to Met230 in human AAR2), is essential for the interaction (Weber et al., 2013).
The low sequence conservation and the resulting structural differences in the AAR2–PRP8RH interface might also have consequences for the wider protein interaction network around AAR2. Concomitant binding of the Prp8p RH and JM domains by Aar2p in yeast sequesters the JM domain, preventing binding of the Brr2p RNA helicase to Aar2p–pre-U5 snRNP (Weber et al., 2013; Galej et al., 2013). In yeast Aar2p–Prp8p complexes (Weber et al., 2011, 2013; Galej et al., 2013), the C-terminal tail of Aar2p runs along the protruding Prp8pRH β-finger module, stringing the β-finger and the central Prp8pJM β-sheet into an extended, intermolecular β-structure (Figs. 1b and 1c).
While the beginning of the C-terminal tail in human AAR2Δloop maintains similar interactions with PRPF8RH as in yeast, for example employing Val373–Val375 to form a short β-sheet of three hydrogen bonds to PRPF8RH, distal parts of the C-terminal tail (beyond Val374) deviate from the direction of the Aar2p C-terminal tail (Figs. 1b and 1c). In yeast, the formation of the penultimate β-strand of Aar2p and the concomitant sequestration of JM from Brr2p is mediated exclusively by a series of hydrophobic residues at the very C-terminus of Aar2p, which are complementary to hydrophobic residues of the neighboring β-strands of RH and JM (Weber et al., 2013; Galej et al., 2013). A structure-based alignment revealed that the respective very C-terminal residues of AAR2, Pro378, Glu379, Gly380 and Glu382, are unlikely to support β-sheet formation with the corresponding highly conserved residues of the PRPF8RH β-finger and PRPF8JM due to their steric or polar properties (Supplementary Fig. S1; compare Figs. 1f and 1g). However, we cannot exclude that in the context of the full-length proteins the very C-terminus of hAAR2 may engage in a yeast-like interaction with the PRPF8 JM domain.
The C-terminal tail of human AAR2Δloop in the observed conformation would not be able to concomitantly bind the PRPF8JM domain as observed in yeast. Indeed, also confirming a prior study (Malinová et al., 2017), AAR2–PRPF8RH did not stably bind PRPF8JM in analytical SEC (Figs. 2g and 2h) and failed to sequester PRPF8JM from a pre-formed SNRNP200395–2136–PRPF8JM complex (Fig. 3a and 3b).
AAR2 alone or in complex with PRPF8RH did not bind stably to SNRNP200395–2136 or to a SNRNP200395–2136–PRPF8JM complex (Figs. 3c and 3d). Instead, a stable AAR2–PRPF8RM-JM–SNRNP200395–2136 ternary complex was formed upon mixing the components (Fig. 3b).
3.3. Conserved Aar2 phosphorylation between humans and yeast
Aar2p can be phosphorylated at five positions in vivo (Ser253, Thr274, Tyr328, Ser331 and Thr345) and phosphomimetic S253D or S253E variants of Aar2p interfered with Aar2p–Prp8p interaction in yeast extracts (Weber et al., 2011). Structural analysis of a phosphomimetic Aar2pS253E variant suggested that phosphorylation leads to a local conformational rearrangement of the Aar2p CTD and thereby to disruption of the Prp8pRH binding site (Weber et al., 2011). A structure-based sequence alignment revealed that Ser284 in human AAR2 corresponds to Ser253 in yeast Aar2p (Supplementary Fig. S1; Fig. 3e), and AAR2 has been found to be phosphorylated at Ser284 in human liver cancer cells (Hornbeck et al., 2012). Recapitulating the situation in yeast, an AAR2S284E phosphomimetic variant failed to stably bind PRPF8RH in analytical SEC (Fig. 3f). Taken together, our interaction studies reveal differences in the relative importance of AAR2/Aar2p regions in maintaining a stable interaction with the PRPF8/Prp8p RH domain in the human and yeast systems. Furthermore, AAR2 does not sequester the PRPF8 JM domain to intermittently prevent SNRNP200 association with the U5 snRNP. AAR2 displacement from PRPF8 may involve reversible phosphorylation of AAR2 at Ser284. Thus, the U5 snRNP assembly steps apparently differ in detail in yeast and humans.
3.4. Human AAR2 counteracts a step 2-like conformation in the PRPF8 RH domain
Mutations in the prpf8 gene can lead to retinitis pigmentosa (RP; Růžičková & Staněk, 2017), a disease that causes blindness in humans, and the corresponding PRPF8/Prp8p variants cause defects in U5 snRNP assembly (Malinová et al., 2017) and splicing (Mayerle & Guthrie, 2016; Mozaffari-Jovin et al., 2013) in humans and yeast. In baker's yeast, two sets of prp8 mutant alleles, corresponding to RP-related mutations in humans that disrupt either the first or the second step of splicing, cluster in the Prp8p RH domain (Grainger & Beggs, 2005).
Furthermore, the human PRPF8 RH domain can undergo a conformational switch in a protruding β-finger module, with one conformation promoting the first step and an alternative, Mg2+-bound conformation supporting the second step of splicing (Schellenberg et al., 2013). Despite the biochemical and structural evidence reported previously, which supports this switch, a caveat of our AAR2–PRPF8RH structure may be that the RH β-finger module makes crystal contacts with a neighboring symmetry-related RH β-finger module. However, recent cryogenic electron-microscopy structures of spliceosomes also confirm this conformational switch, rationalize some of the effects of PRPF8 RH domain variants and demonstrate repeated, long-range repositioning of the PRPF8/Prp8p RH domain during the splicing reaction in yeast and humans (Wan et al., 2016; Bertram, Agafonov, Liu et al., 2017; Yan et al., 2015, 2017; Zhang et al., 2017; Bertram, Agafonov, Dybkov et al., 2017; Rauhut et al., 2016; Galej et al., 2016; Plaschka et al., 2017; Fica et al., 2017; Wilkinson et al., 2021).
Comparison of our AAR2Δloop–PRPF8RH structure with the PRPF8RH step 1 and step 2 conformations revealed that AAR2 binding is compatible with the PRPF8RH step 1 conformation but that steric clashes ensue between the AAR2 C-terminal tail and the PRPF8 RH domain in the Mg2+-bound step 2 conformation (Figs. 4a–4d). To test whether AAR2 is likely to prevent a switch of PRPF8RH into the step 2 conformation, we explored whether AAR2 binds a PRPF8RH variant that is stabilized in the step 2 conformation (T1789P; Schellenberg et al., 2013). Indeed, unlike WT PRPF8RH, PRPF8RH,T1789P partly dissociated from AAR2 upon increasing the Mg2+ concentration in analytical SEC (Figs. 4e–4h), suggesting that a step 2 conformation in PRPF8RH is incompatible with AAR2 binding.
4. Discussion
We have elucidated similarities and differences in the structures and interaction profiles of yeast Aar2p and human AAR2 and have identified a putative, conserved phosphorylation event that is most likely to be involved in the functional cycle of AAR2 as a U5 snRNP assembly factor. Based on our findings, we conclude that the precise roles of Aar2p and AAR2 in U5 snRNP biogenesis differ. In yeast, an Aar2p–pre-U5 snRNP, from which the Brr2p RNA helicase is excluded, seems to constitute an important U5 snRNP assembly intermediate (Boon et al., 2007; Weber et al., 2013). In contrast, our observations of (i) human AAR2 failing to sequester the PRPF8 JM domain from SNRNP200395–2136 and (ii) AAR2 concomitantly binding to a PRPF8 fragment encompassing the RH and JM domains and SNRNP200395–2136 suggest that an equivalent, long-lived intermediate is not formed in the human system. Association of AAR2 with the PRPF8 RH domain as in our AAR2Δloop–PRPF8RH structure would prevent the PRPF8 RH domain from engaging with other regions of PRPF8, the N-terminal region of SNRNP200, the C-terminal region of PRPF31, PRPF6, U4/U6 di-snRNAs and U5 snRNA as observed in the human U4/U6-U5 tri-snRNP (Agafonov et al., 2016; Charenton et al., 2019). This finding suggests that prevention of the premature association of U4/U6 di-snRNP components with pre-U5 particles may be an important function of AAR2 in the human system. In addition, transient blocking of binding sites on the PRPF8 RH domain, possibly supported by allosteric effects due to the selective stabilization of a step 1-like conformation in the PRPF8 RH domain by AAR2, may help to order assembly steps during U5 snRNP biogenesis. The above findings and suggestions are in agreement with the previous observation of the interaction of human AAR2 with a PRPF8–EFTUD2–SNRNP200–SNRNP40 U5 submodule (Malinová et al., 2017).
As most protein-coding genes in humans contain multiple introns (Lee & Rio, 2015), pre-mRNA splicing is an inherent step in their expression. Moreover, pre-mRNA splicing predominantly occurs co-transcriptionally (Alpert et al., 2017) and splicing is physically and functionally coupled to transcription, other pre-mRNA processing steps and mRNA export (Carrocci & Neugebauer, 2019; Tellier et al., 2020). Thus, efficient splicing is a prerequisite for efficient gene expression and, due to its stabiliziation of the step 1 configuration of RH, a potential role of human AAR2 in pre-mRNA splicing cannot be ruled out. AAR2 may have a moonlighting function during pre-mRNA splicing independent of its role as a U5 snRNP assembly factor. By binding the PRPF8 RH domain during a stage of splicing when it is available, for example, in the pre-catalytic B complex (PDB entry 7abg; Townsend et al., 2020), AAR2 may hinder the transition to subsequent stages, thus impeding splicing and, as a consequence, gene expression. As in the case of U5 snRNP assembly, direct blocking of binding sites on PRPF8RH and allosteric effects due to the stabilization of a step 1 conformation in PRPF8RH may support such a splicing-inhibitory role of AAR2. Again, the observed high nuclear levels of AAR2 might ensure that sufficient AAR2 is available to serve multiple functions, as moonlighting is known for some splicing factors that are in excess over other splicing machinery. For example, U1 snRNP has additional roles in 3′-end processing of Pol II transcripts (telescripting; Di et al., 2019). However, AAR2 has never been found to be associated with the spliceosome at any stage of splicing (Agafonov et al., 2011), arguing against a direct effect of AAR2 on splicing. Further studies on human Aar2 in a spliceosomal context will hopefully resolve these remaining questions.
5. Data availability
Structure coordinates and diffraction data have been deposited in the Protein Data Bank (http://www.pdb.org) under accession code 7pjh. All other data supporting the findings of this study are described in the manuscript or in the supporting information or are available from the corresponding authors on request.
6. Related literature
The following references are cited in the supporting information for this article: Barton (1993), Kabsch & Sander (1983) and Pettersen et al. (2004).
Supporting information
Supplementary Figures. DOI: https://doi.org//10.1107/S2059798322009755/cb5141sup1.pdf
‡Present address: 4TEEN4 Pharmaceuticals GmbH, Neuendorfstrasse 15A, 16761 Hennigsdorf, Germany.
§Present address: Berlin Institute for Medical Systems Biology, Hannoversche Strasse 28, 10115 Berlin, Germany.
¶Present address: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, United Kingdom.
We are grateful to Reinhard Lührmann, Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany for the gift of rabbit αAAR2 polyclonal antibody. We acknowledge access to beamlines BL14.1, BL14.2 and BL14.3 of the BESSY II storage ring, Berlin, Germany via the Joint Berlin MX-Laboratory sponsored by the Helmholtz Zentrum Berlin für Materialien und Energie, the Freie Universität Berlin, the Humboldt-Universität zu Berlin, the Max-Delbrück Centrum, the Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Charité – Universitätsmedizin Berlin and the Max-Planck-Institut für Kolloid- und Grenzflächenforschung, Potsdam, Germany and to beamline P14 of the PETRA III storage ring, Deutsches Elektronen-Synchrotron, Hamburg, Germany. Author contributions are as follows. KFS, JA, CH and GW conducted molecular cloning, expression, protein purification and interaction studies. KFS, CH and GW conducted crystallographic analyses. All authors participated in data interpretation. MP, MCW, FH and GW wrote the manuscript, which was revised and approved by all authors. MP and GW initiated the study and directed the project. MCW and FH provided funding for the project. The authors declare no competing interests. Open access funding enabled and organized by Projekt DEAL.
Funding information
This work was funded by a grant from the Deutsche Forschungsgemeinschaft (TRR186/A15 to FH and MCW).
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Cases 1 - 2 of 2
Burt et al v Caraustar Industries Inc. et al
as 4:2022cv11937
Plaintiff: Michele Burt, Christopher Cerasuolo, Nancy Donovan and others
Defendant: Caraustar Industries, Inc., Greif, Inc., The Newark Group, Inc. and others
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Type: Other Statutes › Environmental Matters
UFCW Local 50 Pension Fund et. al. v. RC Wholesale Distributors Inc. et. al.
as 2:2021cv05297
Plaintiff: UFCW Local 50 Pension Fund and Its Trustee
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Type: Labor › Labor: E.R.I.S.A.
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In an attempt to expand into the class action settlement recovery market, Portfolio Recovery Associates, Inc. (PRAA) announced on Monday the acquisition of 62% stake in the Claims Compensation Bureau, LLC (CCB), a company that specializes in recovering funds and processing payments due under class action claims settlement for clients. Although the deal has been completed, the terms of the transaction remains undisclosed.
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