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Greetings again my sexxxy readers! As you may recall from my last month's rant, this magazine had been the target of relentless spamming originating from a politically oppressed and religiously fundamentalist country (NO! Not the USA!). Thanks to the actions of our Editor (the stud muffin Mr. Steve Nelson) and Webmaster Mike (God's gift to the cyber world) we were able to head off the attack.
Of course having a few acquaintances with the Israeli Moussad didn't hurt either (to Avi and Dani: Shalom Guys). I am pleased to announce that the spamming peckerhead is no more. Once again, Karma kills Dogma.
Now on to your questions, as I promised in my last installment, we will address the subject of female ejaculation and anal sex. Together or apart, they are both very enjoyable and logistically possible.
Dr. Corynna C (address redacted) asks,
¨Dear Antonia,
Is it true women can ejaculate? My boyfriend Charles says we can, but I don't believe him
. ¨
My dear Dr. C, where did you get your medical degree from? The mail? The answer is a resounding YES! In fact there are dozens of web sites and videos out there showing the how and wherefore of that phenomena. You and Charles may want to watch ¨Seymour Butt´s Home Movies¨. In his own personable style Seymour demonstrates exactly how to achieve the ultimate female orgasm. Now, for an illustrated example you may want to check out www.squirtqueen.com. Wow! Now that's what I call streaming video. These sight features flash video and live ¨squirt cams¨ you can download. The quality of the pictures is so clear and so graphic I almost felt that I needed to wear goggles. So there you have it, go ahead and check out the information and do try it at home. PS, tell Charles not to forget his goggles.
And now for the back door approach:
Michael Levarre (address redacted) wrote the following:
¨Dear Antonia,
My girlfriend and I are interested in exploring the pleasures of Anal Sex. We want to approach this in a safe way so that she doesn't get hurt. Is there a book or video you would recommend? ¨
Well, I certainly WOULD NOT recommend ¨Last Tango in Paris¨! There is something about Marlon Brando and butter that makes me wanna shout: ¨I can't believe it's not butter!¨ But seriously, I would definitely recommend a video by the Legend of Porn herself: Nina Hartley. Nina has made a very comprehensive documentary entitled ¨Nina Hartley´s guide to Anal Sex¨. In it she gives-no pun intended- a blow by blow account on that forbidden pleasure. This opus is available at Adam and Eve.com
Now this next letter makes me want to coordinate a fundraiser for the sender's benefit. Don't really know how it's done but if you hum a few bars I´ll fake it. Here it goes-
¨He is just a poor boy from a poor family, spare him his life from this monstrosity!¨
Moses Wisdom Bature at (address redacted)@yahoo.com wrote the following....
Permit me to introduce my self. My name is Moses Wisdom Bature. A 22 year old boy from Nigeria.
I come from a poor family which I am the first born and I have five younger.
Please all I am asking is if you can bring me into the film world or make some kind of connection for to some big short in the film and music world. This is a request from a poor boy like who want to make good use of his talent.
Now, everybody together:
¨Scaramouche, Scaramouche, can you do the Fandango?!¨
With apologies to the late Freddie Mercury and Queen.
And speaking of ¨Queen¨ I want every one to get up off their Nina Hartley bottoms and salute the Rainbow Flag as you read this:
Chris Parry at (address redacted)@yahoo.com wrote the following....
Where can I find a list of companies those make/produce gay adult movies?
Chris, my darling transplanted Brit, may I suggest you go out to your local newsstand and get yourself a copy of the ubiquitous Adult Video News. Each month AVN devotes an entire section of its issue exclusively to the Gay Porn Genre. They even offer very comprehensive coverage to the GayVN Awards, which is the Gay version of the prestigious AVN awards. Bottoms up!
And now gentle readers, I would like to give my endorsement to yet another out of this world site. I happened to be contacted by a lovely lady by the name of Anoushka. In her email she had this to say to me:
¨The 20th century gave us many types of sex symbols. From the silent-screen
star of the 20s to the centerfold of the 50s and the supermodel of the 80s,
each sex symbol has left a lasting impression on our culture and the
entertainment that we consume.
My new site, AnoushkaTV.com, presents the first sex symbol of the 21st
Century - the Internet movie star.¨
Folks, in my never ending quest to give the public what it wants; I personally navigated this site and let me just say - Long live The Former Soviet Union! Long Live the Alliance between our 2 great Nations in the never ending struggles to give the proletariat the sexual entertainment it deserves!
To see what all this rhetoric is about, see for yourselves by going to Anoshka´s site at www.anoushkaTV.com and your will be getting down on your knees to thank Comrade Lenin himself giving us the likes of the lovely Anoushka!
There are a few more exceptional sites and webrings worth mentioning; this is a result of the email from the following gentleman:
From: Pete Jenks (address redacted) wrote:
To: firstname.lastname@example.com
I would like to know if it is possible to email contact any exotic
thank you!
Yes Pete there is a Santa Claus; you must go these two web rings. One is www.skymonroe.com which is a portal to other dancers in the ring. The other is www.adultxxx.com both of these web rings offer emails, dates, schedules, and all sorts of hot info that will keep your horney head spinning.
Finally, just what the Doctor ordered.
As my readers may recall, this column and yours truly in particular was the recipient of a ¨Best of the Net Award¨ from About.com the prestigious Internet search engine. It seems that the sexy Dr. Sandor Gardos cannot get enough of us and continues shower us with accolades. He recently wrote this:
¨ We love your site!
so much we placed a link to it on our website, About's Sexuality channel: When we find a site that is particularly outstanding or inspiring, we list it in the relevant subject directory on our About site. Again, congratulations on having such a quality site!¨
Thank you Doc, I´ll take two Cabin Boys and call you in the morning.
So that is all for now my Hot´n´horney readers. Stay tuned to this column where we will discuss such topics as BBW´S, Swingers, and the blossoming on and off screen romance of Jenna Jameson and Brianna Banks. [Story on their next video coming. -Ed.]
In the mean time keep those hot emails cumming!!!
Adult Webcam Conference
Daddy's Girls 2 ~ Reviewer Rated
Calvert Joins Gamelink
Interrogated ~ Reviewer Rated
1st AFFLV Hits Cashman
Richelle Ryan Naked Yoga
Morgan, Lindemulder Headline Exxxotica
A French Documentary Like No Other
Night at the Erotic Museum
Hill and Howard Raw
Taboo is Delicious
Night, Rai, Moore, on Inside
Candida Royalle Passes
Cindy Starfall Appearances
Priya in NYC
More w/Mo Celebrates 100
Angel, Albrite AVN Awards Hosts
The Upward Trending Trannie
Elizabeth Bentley Hits Circuit
Tony Batman to Big Easy
Cleopatra Dead
Introducing Bitcoin Porn Sites
Candidate Sex Tape Scandal
Donald Trump Porn Parody
Savana Styles Available
Sex with Donald Trump
Ashley Sinclair Sex Tape
Mercedes Carrera NightMoves Nom
Allie Haze in Columbus
Vivid NYC Nommed
A Leaner Message
The News Rack: Naked Swingers
I Know You
MWA: 10th Anniversary Column
Tara Lynn Foxx Retires
Taormino Speaks at Goucher
It's Just Work
Tribute to Christian Mann
Testing the Waters
A Legend Looks Back
The Goddess Lives
Resurgence of the Goddess
More on Belle Knox
2014 AVN News Rack Part 2
Belle Knox: Feminist Revealed
Entertainment is Entertainment is Entertainment
Columns Section Index


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Did you know that processing remittance advice is now easier?When considering the further development of the accounting module, we react not only to the legislative changes, but we also listen to wishes and requirements of our customers. The one of many areas that we have decided to enhance is remittance advice.
Inforum 2015 Europe is approachingJoin us on 3rd and 4th November in Paris and attend one of the many conference sessions, for example those for SyteLine. The choice is really huge – from Infor´s products (e.g.
ROSTRA upgrades to Infor CloudSuite Industrial 9ROSTRA s.r.o. has been using Infor SyteLine 7 for many years. In August this year we have jointly initiated the upgrade to the latest version of CloudSuite Industrial (SyteLine) 9. ROSTRA has decided to upgrade for several reasons.

subset: commoncrawl


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Pizza Hut Delivery Drivers job in Canonsburg, PA
- Company
- Pizza Hut
- Job Title
- Delivery Drivers
- Job Type
- Full-time, Part-time
- Hours
- Varies
- Wages
- Competitive
- Location
- 101 Cavasina Drive
Canonsburg, PA 15317
Apply Now
See all Pizza Hut jobs.
You need Adobe Flash Player to see this video!
We have a full-time/part-time opening for a Delivery Drivers.
Must be able to work various shifts per week.
Wage: Competitive
Additional Info
- Driving
- Valid Driver's License


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The Italian Voice Radio Show !
Good Afternoon Everyone,Today on the Italian Voice i want to take this time to Welcome to The Italian Voice my New Co-Host Robin Curasco,.We will be discussing lot's of Interestng Topics that will leave you in stiches and be begging for more,So get to your Laptops & Cellphone's and Log in @Blogtalkradio.com and give us a Listen and or Call in @347-426-3972 and let's hear what you have to say about our topics,.,Not only Serious but Always Hysterical on the Italian Voice,,So Join us where the fun and laughs don't stop and the caller's want to call back every Sunday,,See you on the show,, Today @2pm-3pm..
The Italian Voice Radio Show !
Today i will be broadcasting about the Muslim Kid bringing the Clock to School and getting Rewarded for it,So bring your thoughts to the Show and let's hear what you think,So get to your cellphones and laptops and join in with the Most Honest talk in New Jersey with your host Dom Calvitto only on The Italian Voice Radio Show,Call the Studio @347-426-3972 at 1pm today see ya there,!!
Dudley Enterprises Computer Sales and Repair Saves You Money !
in Technology
For more than thirteen years, Dudley Enterprises Computer Sales and Repair has supplied its customers with quality desktops and laptops. We are a business partner, a trusted provider of Business and Residential Computer Service Contracts backed by a solid guarantee.
Our mission is to listen to our client's specific needs and deliver quality products through continuous operational excellence. We carry complete product lines such as Dell, HP and Lenovo.
Heart2Hearts about the Arts with Aliscia featuring Author Johnny Frost
Author Johnny Frost stops by BRPP Network Radio to discuss with Aliscia Sunshine BlackRose Melton and Ericka Red Dawn Taylor his love for the art of writing and his book
We will also have our new co-host Ericka Red Dawn Taylor in the building.
So hit us by phone line or listen in on your PCs or Laptops for a great show!
Tech Talk with Kingsley Meyer, Mike Thompson and Duane Rigsby
in Training
TECH- TALK with Kingsley Meyer, Mike Thompson, and Duane Rigsby. Our world has made great strides in technology with an ever growing variety of different digital devices. Kingsley, Mike and Duane (each with over 25 years' experience in using, fixing and upgrading,) will discuss different kinds of mobile devices: phones, tablets, and laptops. They will give the pros / cons on how certain devices can best meet the needs of students and those in the working world. They will look at the different operating systems such as Android, iOS, Windows, Kindle Fire HD, and blackberry. Likewise, they will highlight how individuals can acquire different and popular 'apps' and accessories available in today's market. And finally, discussion will surround upfront and hidden costs of owning today's mobile devices.
The Career Expert Live - With Johnstown Tech Support Owner Kevin
in Self Help
We all run into technical problems with our pc's, laptops, and devices. Learn how to handle the most common problems and also how to start your own business in the tech industry from one of our show's sponsors, Johnstown Tech Support. We will be talking to Kevin Masciantonio. Kevin brings many years of experience in solving the most challenging technical problems. With his company, you can be in any part of the world, and they are able to remote in to your pc or laptop and work the problem. This is going to be a great can't miss technical episode.
Saturday Morning Cereal - The "Steal This Episode!" Episode
in Pop Culture
Take some time this week to remember your original happy hour: Saturday mornings as a kid, waking up at dawn, jumping on the couch with a bowl of cereal, turning on the 'toons, tuning out the outside world, and working your way into a sugar hangover before noon. Join us this week as we explore the moral fringes of fandom, or, in other words: To download or not to download, that is the question! In this changing world of digital distribution and cordcutting and rising utility costs, where exactly is the line between a reacting audience member and a jilted consumer? Grim talks to Dr. Chauntelle Tibbals (author of Exposure: A Sociologist Explores Sex, Society, and Adult Entertainment) and Nate Glass (founder of Takedown Piracy), plus the guys remember the blockbusters of 1985 (arguably the Greatest Summer in Cinema, a full THIRTY years ago now!) and how they affect what we're seeing in the theaters (and/or on our laptops) today. You can either listen to it for free on Blogtalk.com and iTunes, or maybe you can steal it from a sketchy Russian site called "www.FreePodcastStuxnetListenNow.com/virus." This is America; it's your choice.
"Family Talk Wednesday" Why We're Failing As Parents
in Family
Our children are becoming more and more exposed to technology and social networkng communities everyday and at such a very early age. iPods, iPads, Androids, iPhones, Laptops, Xbox, Facebook, and more are overwhelming our children on a daily basis and they are becoming more dependent on these gadgets than ever before. In school, work, and home, technology is taking a far greater influence over our children than family values. How can we balance parental control, Internet safety, and healthy lifestyles with such rapidly growing tools when they are being used and given to our children everywhere they go?
Dudley Enterprises will Speak on Cyber Bullying
in Computers
For more than thirteen years, Dudley Enterprises Computer Sales and Repair has supplied its customers with quality desktops and laptops. We are a business partner, a trusted provider of Business and Residential Computer Service Contracts backed by a solid guarantee.
Our mission is to listen to our client's specific needs and deliver quality products through continuous operational excellence. We carry complete product lines such as Dell, HP and Lenovo.
Please bring pen and pad, as these two Georgia entrepernurs Mr.Tyrone Dudley, and his wife Mrs. Cheryl Dudley,aid parents to squash their household 'Back to School computer budget', as well as, trying to help protect and squashing the fastest growing danger for teens, right behind 'Testing While Driving. Its called, 'Cyber-Bulling'!
The Dudleys' are now a part of the best hyper-local news site in the country, OurTown Media. Just listen for now and take good notes Parents
The Tech Duo, will be presenting continuous suggestions, and help correct your computer needs, from time to time. Before you buy five I Phone 16000 , or buy the Windows 4500, parents just listen and help us save ,'One Life'.
Simply call 1-646-478-0188 exactly at 6:00 pm(est), August 6, 2015. You can call from any phone. It will only be 30 minutes, and will able to hear anytime from the host site.
News in the Now
in Technology
"The LaPass RePorT" Tech Tips Tuesdays on News in the Now!
Brought to you by: LaPass Productions
New Media Consulting+
"As technology gets smarter, so do our clients!"
Digital Job Search
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"The LaPass RePorT" Tech Tips Tuesdays on "Digital Job Search"
Visit us: LaPassProductions.com
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IOA, IOC agree to roadmap for India's return to Olympics
Top Stories
- Dadri reminds us how PM Modi bears responsibility for the poison that is being spread
- Kill policemen, never commit suicide: Hardik tells Patel youth
- From Arvind Kejriwal to Rahul Gandhi: Who said what on Dadri lynching
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The crucial joint meeting between the Indian officials and the International Olympic Committee in Lausanne today agreed in principle on a roadmap to bring India back in the Olympic fold.
The meeting, which assumed great importance for India's sporting future, will now work out the exact details and the time frame to get the ban lifted since it was imposed on December 4 last for India's failure to adhere to the Olympic Charter.
A top IOA official, who is part of the delegation, said that the meetings have gone off very well and the combined meeting of IOA, Sports Ministry and IOC officials is now in progress to work out the details.
"It was a good and fruitful meetings so far. We are hopeful of an early solution to the issues. A roadmap is being drawn for India's return to the Olympic fold," the top official told PTI.
The meeting was attended by Sports Minister Jitendra Singh, Sports Secretary P K Deb, Beijing Olympics gold-medallist shooter Abhinav Bindra and Olympian sailor Malav Shroff.
The IOA delegation comprised of Archery Association of India vice-president Tarlochan Singh, Squash and Racket Federation President N Ramachandran, Kayaking and Canoeing chief S Regunathan, Hockey India Secretary General Narinder Batra and Jharkhand Olympic Association head R K Anand.
The run-up to the meeting has been marred by the dramatic pullout of IOA Acting President V K Malhotra and IOC Member in India, Randhir Singh.
The two decided to boycott the meeting after the IOC agreed to include Batra and Anand in the IOA delegation for the meeting, in addition to the four-member list submitted earlier.
It is also learnt that the Olympic Council of Asia, which had only yesterday asked the IOC not to interact with the suspended IOA officials and postpone the meeting to mid June,
... contd.


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Off-Campus Dining:
It's average, the typical chain and fast food restaurants, so just normal.
The school has stricter policies since it is a private Christian university, but as a conservative student, I like the strictness because it helps me feel more comfortable knowing that I won't be overly exposed to crazy parties, drinking, and stuff like that.
I was grateful to receive some scholarship money from the school, my sister did as well. They offer some great benefits the first semester if you submit your deposit by a certain date.
Party Scene:
I live off campus, and I don't party anyway. I'm assuming that there is limited partying because it is a Christian private university.
Campus Food:
I have heard mixed opinions about the food. I live off campus, so I really only go to the Starbucks on campus, and not the other food locations.
Campus Housing:
I live off campus so I cannot accurately rate on-campus living. I have however seen one dorm, which was very small, and eaten at one place on campus, which was not the best, but again, I cannot base my opinion on so little evidence.
Campus Food:
The food is excellent with an affordable price!
Campus Housing:
Libert University has a phenomenal atmosphere and great courses to offer. It is a fantastic school. For a private school it is not too expensive! The food is great and the professors really do care about their students. The living spaces are outstanding as well! The dorms are very conveniently located and offer great living conditions.
Campus Food:
I love eating at the Rot, the food there is always great and there are so many choices you are never limited. I also like how they always have fresh fruits and vegetables.
Campus Housing:
The housing is way overpriced and the campus would have many more students living on campus if they reduced the price by even 1,000 dollars.
Liberty has a big fan support system. Everyone loves going to events on the weekends to cheer on the flames.
Off-Campus Housing:
While living off campus is more comfortable, you are take away from the social atmosphere of college. I like live on campus because of the friends that I meet.
Off-Campus Dining:
There is a large variety of off-campus dining options. It makes going off campus for some food a real treat. Most of the variety is real close to school which is nice!
Many believe that our school has too many rules. However, when one looks at the specific rules Liberty has in place, each are set in place to protect the well-being of students.
Though I don't have enough financial aid to cover my schooling, Liberty does provide great financial aid. Keep your grades up because there is an academic scholarship. Also, being on student leadership usually aids in scholarship monies.
Party Scene:
Since drinking is not permitted, parties that take place are always enjoyable and safe. Students can actually remember what they did on a Friday night!
Campus Food:
Though it is not home cooked food, our dining options range from a variety of things allowing us students to get well balanced meals. I like the variety given at each meal time. It ensures that almost everyone will be able to find something!
Campus Housing:
The hall set up ensures that friends will be made
Campus Food:
College food, what is there to say?
Off-Campus Dining:
there are a lot of options of places to choose from.
I have only had to deal with it once so far, but the only experience I have had so far has been a pain but it eventually got worked out on time.
Campus Food:
I absolutely love the mini rot because it is real food and the best part, is that it is small. I do not like the main rot only because it is way to large and I do not do well with a lot of people.
Campus Housing:
I love living on campus because I am surrounded by friends and people that care about me, and me them. Being on campus, I am also close to everything.
Off-Campus Dining:
We have a huge variety of food places around campus.
Some things are strict others are not as strict.
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microRNAs (miRNAs) are extensively involved in developmental programming. Some miRNAs are highly conserved, while others are lineage specific. All miRNAs maturate through a series of processing steps. Here we review recent progresses in the studies of early steps in miRNA biogenesis, focusing on animal systems. The miRNA maturation pathways are surprisingly diverse, involving transcription by RNA polymerase II or III, cleavage by the Drosha nuclease or the spliceosome, and sometimes modifications by the adenosine deaminase ADAR. The relationship between the diversity in miRNA biogenesis and the apparently rapid evolution of miRNA genes and functions is discussed.
miRNAs are a class of non-protein-coding RNAs that are ~22nt in length. They are involved in important biological functions, such as development and cell physiology [1]. Over 700 human miRNAs have been cloned [2]. They regulate the expression of ~30% of protein-coding genes by targeting specific messenger RNAs for cleavage or translational regulation, in a combinatorial fashion [3]. In animals, miRNAs often lead to translational repression, and to some extent mRNA decay, through partially complementary pairing to the 3′ untranslated regions (UTRs) of their targets using the seed region (positions 2 to 7 or 8 from 5′ end). In plants, miRNAs show perfect complementarity to their targets and lead to mRNA degradation. A recent article showed that translational inhibition is also widely effective in gene silencing by plant miRNAs and siRNAs, drawing more similarities between animal and plant miRNAs [4]. Some miRNAs have been shown to be oncogenic, or suppress tumor growth [5]. Several miRNAs are involved in metastasis [6, 7]. Artificial miRNAs that mimic natural pri- or pre-miRNAs (termed small hairpin RNAs, or shRNAs) are being used as a tool for genomic research and for therapeutic purposes [8–10]. The mechanisms of posttranscriptional regulation by miRNAs are nicely reviewed in [3]. In this review, we will focus on recent studies of the early steps in miRNA biogenesis pathways in animals, and discuss their implications in the evolution of miRNA genes and functions.
miRNAs contain 5′-phosphate and 3′-OH, thus they can be cloned using a specific procedure involving size selection and adaptor ligations [11–14]. Most miRNAs are named miR-#, where # represents a number. miRNA sequences are found in either the 5′ or 3′ strand in a hairpin secondary structure of their precursors. If the mature miRNA is located on the 5′ strand, it is called miR-#-5p; if it is located on the 3′ strand it is called miR-#-3p. These common features of miRNAs are defined by miRNA processing factors, as will be discussed later. miRNAs have similar length and chemical structures to another class of small RNAs, called small interfering RNAs (siRNAs) [15]. siRNAs are generated from long double-stranded RNAs, which could be replication intermediates of viruses or transcripts of transposable elements.
miRNAs are synthesized in cells as long primary transcripts (pri-miRNAs) that often contain thousands of nucleotides [16]. Pri-miRNAs are cleaved by a series of cellular processing factors. The major miRNA processing pathway in animals is illustrated in Fig. 1. A long pri-miRNA is first recognized and cleaved by a ribonuclease III (RNase III), called Drosha, along with an RNA-binding protein DGCR8 or Pasha (Partner of Drosha)[17–21]. Jointly, Drosha and DGCR8 form the Microprocessor complex [18, 19]. The product of the first cleavage event, pre-miRNA with ~70 nt, is then transported across the nuclear membrane by Exportin-5 and Ran-GTP[22, 23].
In the cytoplasm, the pre-miRNA is further cleaved at a pair of sites close to the hairpin loop by another RNase III enzyme Dicer to give a miRNA duplex[24–29]. The miRNA duplex is then incorporated into the effector complex RISC (RNA induced silencing complex)[30–32], likely through the activity of the RISC loading complex, which contains Dicer, the central RISC component Argonaute and RNA-binding proteins (TRBP and PACT [33–37] in humans, and loquacious [38–40] in flies). A miRNA duplex is unwound into the mature single-stranded form (called the guide strand) [41] and its complementary strand (the passenger strand or miRNA*) is degraded by RISC[33, 42–44]. Mature miRNAs often predominantly originate from one strand of the duplex. Asymmetric incorporation of siRNA strands has been observed [45, 46]. The Drosophila R2D2, the dsRNA binding partner of Dicer-2, binds the end of the duplex with more stable base pairing interaction, providing the biochemical basis for preferential use of siRNA strands [47]. A similar mechanism might be responsible for miRNA strand selection. The stem loop in pre-miRNAs, presumably when bound to the RISC loading complex, likely contributes to the strand selection [48]. The miRNA* also has a chance to be incorporated into RISC and can be actively used in gene regulation [49].
The extensive processing of miRNA transcripts ensures that only RNA molecules with the proper structural features can be used in gene regulation [50]. These structural features are recognized by the miRNA processing factors. For example, it has been shown that Dicer cleaves double-stranded RNA into 21-nt duplexes preferentially from the end of the helix [51, 52]. In addition, the stem and 3′ overhang of pre-miRNAs are important for association with Exportin-5 and Ran-GTP[53]. The recognition of pri-miRNAs by Drosha and DGCR8 is the first step in miRNA maturation, and probably contains the most important constraints for miRNA processing. The RNA structures within an 80-nt hairpin region and the nonstructured regions flanking the hairpin have been shown to be important for processing by Microprocessor [17, 54–56]. The Drosha cleavage sites are about 10-bp from the junction between the miRNA hairpin and the flanking regions [57]. It is agreed that DGCR8 plays a major role in the recognition of pri-miRNAs [57–59]. However, it is far from clear how DGCR8 and Drosha distinguish pri-miRNAs from the large number of other hairpin-containing RNA molecules. In addition, the DEAD-box RNA helicases p68 and p72, which are found in the Microprocessor complex [19], are required for processing of a subset of miRNAs [60]. Their helicase activity is required for the maturation of these miRNAs and the subsequent gene silencing [60, 61]. These helicases may bind pri-miRNAs and modulate the activity of the Microprocessor complex through structural reorganization [60], or facilitate the loading of miRNA duplexes into RISC [19]. siRNAs are also generated through cleavage by Dicer [24]. Mammals only have one Dicer gene: knockout or knockdown of Dicer affects both miRNAs and siRNAs [62, 63], whereas knockdown of Drosha or DGCR8 only affects miRNA maturation [64–67].
Analysis of the genomic locations of miRNA genes indicated that many miRNAs are located in the introns of mRNAs, with some even in the coding regions [68]. A co-transcription of a miRNA with its host gene presumably allows them to be regulated together. Interestingly, a recent report showed that miR-21 is located in an intron of a coding gene, TMEM49; but its transcription is likely driven by its own promoter [69]. Thus, location of a miRNA in an intron does not warrant co-regulation with its host gene. Other miRNAs are in the intergenic regions and are likely transcribed independently. Stems of miRNA hairpins tend to be conserved; the variation of the loop sequences is increased; and there is a sharp decrease in conservation in sequences immediately flanking the hairpin [70]. This type of phylogenetic shadowing profile was successfully used to identify novel miRNAs in primates. There is a tendency in miRNAs to cluster together [11, 12, 31]. Clustering of miRNA hairpins presumably simplified their transcription regulation. Several recent advances in the study of miRNA biogenesis further underscore the diverse origin of miRNAs and will be discussed below.
Most pri-miRNAs are likely products of the RNA polymerase II [71, 72]. miRNAs regulate the expression of genes through pairing interactions with mRNAs, especially through their 3′ untranslated regions. Therefore, the expression of miRNAs must be regulated. Since transcription by RNA polymerase II is subjected to the highest degree of regulation out of the three RNA polymerase families, it is not surprising that most pri-miRNAs are transcribed by RNA polymerase II. While it makes sense most pri-miRNAs are transcripts of RNA polymerase II, there is no reason to exclude other RNA polymerases to be used. In fact, RNA polymerase III promoters such as U6 and H1 are often used to transcribe shRNAs [8]. In 2006, it was reported that ~50 human miRNAs are transcribed by RNA polymerase III [73]. In a genomic analysis, the miRNAs in the human chromosome 19 miRNA cluster (C19MC) were found to be dispersed among Alu repeats. Alu sequence is the most abundant transposable element in the human genome. It is derived from the 7SL RNA gene, which encodes the RNA component of the signal recognition particle that functions in protein synthesis. The Alu sequence contains the 7SL promoter, an RNA polymerase III promoter. Subsequent chromatin immunoprecipitation and cell-free transcription assays confirmed that the miRNAs in C19MC are indeed products of RNA polymerase III.
Many miRNAs are located within introns. They are in general cleaved by Drosha, parallel to the splicing of their host mRNAs [74]. Deep sequencing of D. melanogaster and C. elegans RNAs allowed the identification of a novel class of intronic miRNAs that do not contain the 10-bp helix at the base of the miRNA hairpin normally required for Drosha cleavage [75, 76]. These pre-miRNAs (mirtrons) turned out to be processed directly by the spliceosome, instead of Drosha (Fig. 1). Like other introns, mirtrons are flanked by 5′ and 3′ splicing sites, and contain branch point sequences. Unlike the pre-miRNAs generated by Drosha, the lariat mirtrons generated by the spliceosome need to be linearized by the debranching enzyme and fold into hairpins, prior to exportation to the cytoplasm by Exportin-5. Both Drosha-processed pre-miRNAs and mirtrons are processed by Dicer. It should be noted that mirtrons are a subset of the miRNAs transcribed by RNA polymerase II.
Pre-miRNAs are typically 60–100 nt in length. This size range coincides with the size of small introns in flies and nematodes. Mammalian introns are usually much longer than pre-miRNAs, thus fewer mirtrons were predicted to exist in mammalian genomes [75]. However, a recent report demonstrated that mirtrons appeared to be present in human and other mammalian genomes [77]. Although several mirtrons are highly conserved within Drosophilids, nematodes and mammals, no mirtron is collectively shared by these animals, suggesting that mirtrons are acquired independently during evolution of different animal clades.
Cleavage by Drosha and Dicer are not the only RNA processing events that miRNAs can go through during maturation. ADAR (adenosine deaminase that acts on RNA) can convert some adenosines (A) to inosine (I) in double stranded RNAs (Fig. 1). I prefers to base-pair with C. The A→I modification disrupts a stable A:U base pair and creates a less stable I:U mismatch. ADAR is the most common type of RNA editing enzyme in metazoans [78]. Editing of pri-miR-142, suppresses its processing by Drosha [79]. Pri-miR-151 and possibly pre-miR-151 are modified by ADARs at two sites close to the Dicer cleavage sites, and these modifications block cleavage by Dicer [80]. Thus, the A-to-I editing could regulate miRNA biogenesis. Interestingly, an A-to-I editing site of miR-376 is located in the "seed" region critical for the recognition of miRNA targets [81]. This modification redirects miR-376 to silence a different set of genes. Therefore, target redirection through ADAR activity can increase the functional diversity of miRNAs. In addition to adenosine deamination, recent deep sequencing of miRNAs extracted from human and rodent showed addition of a single nucleotide at the 3′-end of the miRNAs [82, 83] and cytosine deamination [83]. The functional importance of these modifications remains to be demonstrated.
miRNAs can be generated in animals through transcription by either RNA polymerase II or III; as independent transcripts, or together with other genes; from introns or exons. Their precursors can be processed by either Drosha or the spliceosome in the nucleus and be modified by RNA editing enzymes. The diverse pathways to generate functional small non-coding RNAs are further highlighted by miRNAs in plants [84]. Plant genomes do not encode Drosha and DGCR8 homologues; instead miRNAs are processed by the Dicer-like proteins. Unlike their animal counterparts, plant miRNAs are methylated at their 3′ ends through the activity of HEN1. Therefore, the consensus for miRNA biogenesis is: there's more than one way to skin a cat. As long as the mature miRNAs have the chemical structure and the length required for interaction with RISC and for subsequent gene regulation, it is probably advantageous to have multiple ways to generate them.
Obviously, the distinct pathways allow miRNAs to be controlled through different mechanisms [85]. For example, the Lin-28 RNA-binding protein specifically associates with the let-7 family pri-miRNAs and blocks their processing by Drosha and Dicer [86]. The SMAD proteins, a TGF-β and BMP signal transducers, promote the processing of pri-miR-21 by Drosha [87]. hnRNP A1, a nucleocytoplasmic shuttling protein, binds specifically to human pri-miR-18a and facilitates its Drosha-mediated processing[88]. We found that the DGCR8 protein binds heme and this interaction may be part of a molecular mechanism that regulates miRNA maturation [58]. ADAR editing of pri-and/or pre-miRNAs allows tissue-specific regulation of their processing and target specificity [79–81]. The presence of multiple miRNA maturation pathways predicts that altering the expression or activity of miRNA processing factors changes the abundance of only a subset of miRNAs.
It is possible that distinct pathways used to generate miRNAs allow these small gene regulators to evolve more readily, thus conferring fitness to their hosts. It was suggested that the mirtron pathway may have helped the emergence of miRNAs before the advent of Drosha [75]. When deep sequencing of miRNAs from three species of Drosophila was performed, a large class of evolutionarily young miRNAs was observed [89]. The estimated birth rate of new miRNA genes is 12 per million years. Among the new genes, 96% disappeared quickly in the course of evolution, 4% were retained, and only 2.5% became modestly or highly expressed. Furthermore, sequencing and comparative analysis of the C19MC in nine diverse primate species suggested an Alu-mediated rapid expansion of this miRNA family [90]. Single nucleotide polymorphisms (SNPs), the most abundant form of DNA variation in the human genome, are found in miRNA genes and may provide a snapshot of miRNAs during evolution [91]. Some miRNA SNPs have been shown to alter miRNA processing and target specificity [92, 93]. Editing by ADAR allows miRNA isoforms with different target specificity to be generated from a single miRNA gene [81]. The emergence of new miRNAs correlates with the introduction of developmental complexity during evolution, suggesting important functional contributions from the expansion of miRNA genes, as well as other non-protein-coding RNAs [94, 95]. Large numbers of miRNA-like hairpin structures [96, 97] and mirtron-sized introns [75–77] found in animal genomes could provide potential novel miRNA candidates to facilitate the fast evolution of miRNAs.
This work is supported by NIH grant GM080563-01A1 to F.G.
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