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The 3’UTR of the orb2 gene encoding the Drosophila CPEB translation factor plays a critical role in spermatogenesis

CPEB proteins are conserved translation regulators involved in multiple biological processes. One of these proteins in Drosophila, Orb2, is a principal player in spermatogenesis. It is required for meiosis and spermatid differentiation. During the later process orb2 mRNAs and proteins are localized within the developing spermatid. To evaluate the role of orb2 mRNA 3’UTR in spermatogenesis, we used the CRISPR/Cas9 system to generate a deletion of the orb2 3’UTR, orb2R. This deletion disrupts the process of spermatid differentiation, but has no apparent effect on meiosis. While this deletion appears to destabilize the orb2 mRNA and reduce the levels of Orb2 protein, this is not the primary cause of the differentiation defects. Instead, differentiation appears to be disrupted because orb2 mRNAs and proteins are not properly localized within the differentiating spermatids. Other transcripts and proteins involved in spermatogenesis are also mislocalized in orb2R spermatids. Author summary The conserved family of cytoplasmic polyadenylation element binding (CPEB) proteins can activate or repress translation of target mRNAs, depending on the specific biological context, through interaction with special cytoplasmic polyadenylation element (CPE) sequences. These proteins function mainly in highly polarized cells. Orb2, one of the two Drosophila melanogaster CPEB proteins, is predominantly expressed in the testes and is crucial for spermatogenesis. The 3’UTR of orb2 transcript contains multiple CPE-like motifs, which is indicative of orb2 self-regulation. We have generated a deletion that removes the greater portion of 3’UTR. While this deletion causes a reduction in the levels of orb2 mRNA and the protein, this does not appear to be responsible for the defects in spermatogenesis observed in the deletion mutant. Instead, it is the mislocalization of the mRNA and protein in the developing spermatids.


Introduction
4 75 by meiosis II. At the end of meiosis, a cyst consisting of 64 interconnected spermatids with haploid 76 nuclei is formed. The cells in the cyst remain undifferentiated during the meiotic divisions but start to 77 differentiate as soon as meiosis is completed. One of the first steps is the reorganization and oriented 78 polarization of the germ cells in the cyst so that all nuclei are clustered towards its basal side (relative to 79 the apical basal polarity of the testes). Basal bodies, which function as microtubule nucleation centers, 80 are located on the apical side of the nuclei. They initiate the assembly of the flagellar axonemes that 81 grow towards the apical tip of the testes. The axonemes elongate until they almost reach this tip and then 82 normally cease to grow [19][20][21][22]. During the elongation phase, orb2 transcript and protein are 64-cell cyst is properly polarized, orb2 contributes to the elongation of the flagellar axonemes. It is 102 required for localizing transcripts in a comet pattern in the growing flagellar axonemes and activating 103 their translation. The transcripts whose localization and translation depend on Orb2 include orb2 and 104 apkc mRNAs [18,27]. orb2 mutants are also defective in that the growth of the flagellar axonemes is 105 not properly terminated and defective ICs are assembled.

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Studies on the other fly CPEB protein, Orb, have shown that it has a positive autoregulatory activity:

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Orb binds to sequences in the 3'UTR of its own transcript, localizes the transcript to the developing 108 oocyte, and controls its on-site translation. This 3'UTR-dependent autoregulatory activity helps drive 109 oocyte specification in the newly formed 16-cell cysts, while at later stages of oogenesis it is important 110 for ensuring that sufficient levels of Orb protein are localized to the developing oocyte [14,28]. Similar 111 to the orb 3'UTR, most of the orb2 3'UTRs are quite long and carry multiple CPE-like elements.

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Moreover, we and other authors have found that Orb2 is associated with orb2 transcript in vivo [18,29,113 30]. Hence, the question has arisen whether the orb2 3'UTR has important functions in 114 spermatogenesis. To address this question, we used the CRISPR/Cas9 system to delete the orb2 3'UTR 115 and analyzed the mutants for the effect of this deletion on spermatogenesis.

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Deletion of the orb2 3'UTR 119 The orb2 gene is predicted to encode five transcript species that differ in their transcription start sites, 120 splicing patterns, and the lengths of their 3'UTRs. Only one of them, RA, encodes the smaller 60-kDa 121 Orb2A isoform. As shown in Fig 1, RA  transcripts, RD and RH, have 3'UTRs of 3826 and 5791 nt, respectively. The distribution of canonical 127 CPEs in these transcripts is different. There is one canonical and six non-canonical CPEs in the 1563-nt 128 RC 3'UTR, whereas the two larger 3'UTRs have 27 and 37 CPE-like sequences respectively that 129 include 9 canonical UUUUAU motifs.

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As the RD and RH 3'UTRs contain a much greater number of canonical and non-canonical CPEs, we 131 designed a deletion that selectively removes the bulk of their 3'UTR sequences. As shown in Fig 1,  RH. For all three of these transcripts, the 3'UTR sequence upstream of the deletion breakpoint is 1008 138 nt long and contains five non-canonical but Orb2-enriched CPEs (UUUUUGT or UUUUUGUU).

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In the initial fly stock, the deleted DNA was replaced by the DsRed gene flanked by loxP sites and an 140 attP sequence. DsRed was then excised to give the orb2 gene carrying attP and loxP sites in place of the 141 4522-nt deletion. The deletion was verified by sequencing, and the resulting mutation was designated 142 orb2 R .

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Most orb2 R males are sterile 144 While orb2 null alleles are semi-lethal, with only a few flies surviving to adulthood, the orb2 R mutation 145 does not appear to affect any essential processes during development, since the number of homozygous 146 orb2 R flies reaching the adult stage is close to that in wild-type (WT) flies (Suppl. Fig 1). On the other 147 hand, male fertility in the orb2 R mutants is reduced. Figure 2 shows the results of experiment where 35 148 orb2 R males were mated with 70 WT females. After 10-day incubation, adult flies were removed from 149 the vials and their offspring were allowed to develop to adulthood. Quantification of the number of 150 offspring indicates that the overall fertility of orb2 R males is reduced approximately tenfold. 7 151 Two scenarios could potentially explain the reduction of male fertility in orb2 R flies. First, the 152 production of functional sperm could be more or less uniformly impaired in all orb2 R males. An 153 alternative, though seemingly less likely possibility is that the fertility of individual flies could be 154 affected differentially, so that some males are fertile while others are sterile. To distinguish between 155 these alternatives, we measured the fertility of individual orb2 R males. In the first experiment, we mated 156 individual males to two WT virgin females for one week and then scored the number of males that 157 produced offspring. Unexpectedly, we found that second scenario was correct: about 75% of the orb2 R 158 males were completely sterile (Fig. 2B). When orb2 R was placed in trans to the null allele orb2 36 (a 159 deletion of orb2), no offspring were produced. Moreover, the number of offspring from the few fertile 160 orb2 R males is substantially reduced, compared to WT or to males heterozygous for orb2 36 . In the 161 experiment shown in Fig. 2C, we mated males of the above genotypes to WT females and then scored 162 the number of offspring they produced. WT males typically have more than 60 offspring, while males 163 heterozygous for an orb2 null allele, orb2 36 , have slightly fewer, with an average of a bit more than 60 164 offspring. In contrast, of the fertile orb2 R males most had substantially reduced fertility and produced 165 fewer than 30 offspring.

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The accumulation of orb2 transcript and protein is reduced in orb2 R testes 167 To better understand the nature of spermatogenesis defects in the orb2 R mutant, we examined the and orb2 R males. The GADPH transcript, which lacks canonical CPEs, served as a control for RNA 171 input. As shown in Fig. 3A, the level of orb2 mRNA in orb2 R testes is reduced approximately by half, 172 compared to WT. This appears to be due to the decreased stability of the mRNA, since the level of orb2 173 primary transcripts (detected with primers located on the intron-exon junction) in orb2 R testes is close to 174 that in WT.

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Both Orb2 isoforms were detected in Western blots of extracts from orb2 R testes, but the 75-kDa 176 isoform in mutants proved to be reduced, compared to WT. The difference in the levels of this protein is 8 177 illustrated in the blot of serial dilutions of the extracts from WT and orb2 R males (Fig. 3B). Quantitative 178 analysis ( Fig. 3C) showed that the 75-kDa isoform of Orb2 in mutant males was reduced approximately 179 twofold, as in the case of orb2 mRNA.

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The above experiments indicate that the deletion of sequences in the 3'UTRs of the orb2 transcripts 181 results in a twofold reduction in both mRNA and protein levels, while transcription is unaffected. This 182 finding suggests that the deletion mutant mRNAs are less stable. However, it is surprising that a two-183 fold reduction in the level of mRNA and protein is sufficient to significantly perturb spermatogenesis so 184 that most orb2 R males were sterile. In fact, there is no evidence of a strong haploinsufficiency as males 185 heterozygous for the orb2 deletion (orb2 36 ) produced nearly as many offspring as WT males (Fig. 2C) 186 and showed no obvious abnormalities in spermatogenesis. To confirm that orb2 36 /+ testes have the 187 expected two-fold reduction in orb2 gene products, we compared orb2 mRNA and protein levels in WT 188 and orb2 36 /+. As shown in Fig. 3A, the amount of orb2 mRNA in orb2 36 /+ testes was only about a 189 quarter that in WT. By contrasts, orb2 mRNA was only reduced about two-fold in orb2 R . Similar results

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were obtained when we compared protein levels in WT and either orb2 36 /+ or orb2 R /orb2 36 . The level 191 of Orb2 protein in orb2 36 /+ was about one third that in WT, and about one quarter that in orb2 R /orb2 36 . 192 These findings indicate that the reduction in orb2 mRNA and protein in homozygous orb2 R testes is if 193 anything less than that in orb2 36 heterozygotes, while the fertility of these flies is significantly different.

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Hence, it is unlikely that the reduction in mRNA and protein in orb2 R testes is in itself responsible for 195 the significantly reduced fertility of the 3'UTR deletion mutant.

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The orb2 3'UTR is required for proper orb2 transcript and protein localization 197 To better understand why most orb2 R males are sterile, we examined orb2 transcript and protein 198 expression during spermatogenesis. The pattern of their accumulation in premeiotic and meiotic cysts is 199 similar to that in WT. The expression of orb2 transcript and protein in orb2 R testes is upregulated after 200 the formation of the 16-cell spermatocyte cysts, and the protein is distributed more or less evenly 201 throughout the cytoplasm. However, the amounts of transcript and protein are reduced, compared to WT 9 202 (Suppl. Fig. 2). Unlike in flies homozygous for the null-allele orb2 36 , spermatogenesis in orb2 R does not 203 arrest prior to meiosis I even though Orb2 protein level are lower than in WT; instead, both meiotic 204 divisions appear to be normal, and 64-cell cysts are formed.

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While spermatogenesis appears to be unaffected through the completion of meiosis, a series of 206 abnormalities become evident once the spermatids begin to differentiate. When they are first formed, the  leaf, early canoe, late canoe, and finally needle stage [19,21,22]. Along with these morphological 254 changes, the nuclei coalesce into a tight bundle to form an inverted cap-like structure (Fig. 8A) [22]. In 255 orb2 R testes, most of the nuclei in the cysts appear to progress to the needle stage, but their subsequent 256 coalescence into the cap-like structure is defective, with only a few exceptions (~10% of the testes) 257 (Figs 8A, 8C). In about 45% of the testes, only a subset of the spermatid cysts has nuclei that coalesced 258 into a cap-like structure, while in other cyst the nuclei are scattered or display only partial coalescence.

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No coalesced nuclei were found in the remaining testes (~45%) (Fig. 8C). When orb2 R is trans to    Our results indicate that sequences in the orb2 3'UTR have important roles in spermatid differentiation, 314 but they also raise several interesting questions. We have found that the levels of orb2 transcript and 315 protein in orb2 R are reduced about twofold. The simplest interpretation of this result is that the presence 316 of intact 3'UTRs is important for orb2 mRNA stability. However, in view of the sequence organization 317 of the orb2 R deletion, it is also possible that the reduced amount mRNA and, consequently, protein 318 levels is due to an inefficient use of the RH polyadenylation sequence. The approximately twofold 319 reduction in the levels of orb2 R gene products is accompanied by variable and incompletely penetrant 320 effects on spermatogenesis and male fertility. One explanation for these phenotypes is that orb2 is 321 haploinsufficient for several critical steps in spermatid differentiation and maturation. However, 322 heterozygosity for orb2 36 (an orb2 deletion) results in a similar, if not greater, reduction in orb2 323 transcripts and proteins without any concomitant effect on spermatogenesis or male fertility. Thus, a 324 more likely explanation for the impairment of spermatogenesis and fertility in orb2 R is that the deleted 325 3'UTR sequences are required not only for normal mRNA accumulation but also for orb2 function.        Quantification and statistical analysis 425 The orb2 mRNA and protein enrichment was calculated using average intensity projections of the 426 growing end of spermatid cysts (Fig. 4C). First, the mean fluorescence intensity of the growing tip of 18 427 the flagellar axoneme was determined by averaging several z-stacks in the three areas of interest. The 428 mean fluorescence intensity in spermatocytes was determined in the same way. Then the mean 429 fluorescence intensity of each area of interest within a spermatid was divided by the mean fluorescence 430 intensity of spermatocytes for each testis. These ratios are shown as box plots. The Imaris software was 431 used to quantify the fluorescence signal of orb2 mRNA and Orb2 protein.

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Experimental data were processed statistically with the GraphPad Prism software. The statistical 433 significance of the observed differences was estimated by unpaired two-tailed t-test (Figs. 3A, 4C). 434 Mendelian inheritance in the offspring was analyzed using the nonparametric chi-square method.