Coskun, P. et al. (2019) International Worm Meeting "3' UTR mediated translational regulation of glp-1 in the germline of Caenorhabditis elegans."

Coskun, P., Ryder, S.

[International Worm Meeting, 2019]

Translational control of maternal mRNAs is a major form of gene regulation during germline development and embryogenesis. In Caenorhabditis elegans, the maternal gene glp-1 encodes a homolog of the Notch transmembrane protein required for cell proliferation in the germline, and cell fate specification in the embryo. The RNA binding proteins POS-1 and GLD-1 directly regulate the translation of GLP-1 protein by binding to the specific elements within the glp-1 3' untranslated region (3' UTR). When POS-1 or GLD-1 binding is disrupted by mutation of their respective elements, the expression pattern of a glp-1 3' UTR transgene changes in both the germline and in embryos. The mechanism by which POS-1 and GLD-1 mediate translation repression is not well understood. Previous work showed that loss of POS-1 reduces the average polyA tail length of endogenous glp-1 transcripts in embryos. Here, we show that mutation of either the GLD-1 or POS-1 binding motifs in transgenic reporters does not change polyA site selection, and has no effect on polyA tail length distribution, in young adults. These results rule out alternative polyA site usage as a mechanism of regulation, and suggest that the decrease previously observed in endogenous glp-1 polyA tail lengths upon POS-1 RNAi might be indirect. We will expand our analysis by measuring reporter mRNA abundance, polysome association, and polyA tail length distribution in embryos and young adults. When complete, our studies will provide insights of into the mechanism of glp-1 translational regulation in C. elegans germline.

Coskun, Peren et al. (2021) International Worm Meeting "3' UTR mediated post-transcriptional regulation of glp-1 in the germline of Caenorhabditis elegans"

Ryder, Sean, Coskun, Peren

[International Worm Meeting, 2021]

Translational control of maternal mRNAs is a major form of gene regulation during germline development and embryogenesis. In Caenorhabditis elegans, the maternal gene glp-1 encodes a homolog of the Notch transmembrane protein required for cell proliferation in the germline and cell fate specification in the embryo. The RNA binding proteins POS-1 and GLD-1 directly regulate the translation of GLP-1 protein by binding to the specific elements within the glp-1 3' untranslated region (3' UTR). When POS-1 or GLD-1 binding is disrupted by mutation of their respective elements, the expression pattern of a glp-1 3' UTR transgene changes in both the germline and in embryos. The mechanism by which POS-1 and GLD-1 mediate translation repression is not well understood. Previous work showed that loss of pos-1 increases the average polyA tail length of endogenous glp-1 transcripts in embryos. Here, we show that mutation of either the GLD-1 or POS-1 binding motifs in transgenic reporters does not change polyA site selection. This result rules out alternative polyA site usage as a mechanism of regulation. We also show that wild-type glp-1 transgenic reporter embryos have a shorter average polyA tail length compared to mutant transgenic reporters in GLD-1 or POS-1 binding motifs. We further used our reporters to measure the effect of cytoplasmic polyA polymerases, deadenylases and translation initiation factors on the glp-1 expression level and pattern. The results reveal that POS-1 and GLD-1 exert their effects through different pathways. Our studies provide insights of into the mechanism of glp-1 post-transcriptional regulation in the germline and embryo.

Haenni S et al. (2012) Nucleic Acids Res "Analysis of C. elegans intestinal gene expression and polyadenylation by fluorescence-activated nuclei sorting and 3'-end-seq."

Mellor J, Tian B, Eberhard R, Furger A, Hengartner MO, Browne C, Haenni S, Rust N, Hoque M, Sharpe H, Ji Z

[Nucleic Acids Res, 2012]

Despite the many advantages of Caenorhabditis elegans, biochemical approaches to study tissue-specific gene expression in post-embryonic stages are challenging. Here, we report a novel experimental approach for efficient determination of tissue-specific transcriptomes involving the rapid release and purification of nuclei from major tissues of post-embryonic animals by fluorescence-activated nuclei sorting (FANS), followed by deep sequencing of linearly amplified 3'-end regions of transcripts (3'-end-seq). We employed these approaches to compile the transcriptome of the developed C. elegans intestine and used this to analyse tissue-specific cleavage and polyadenylation. In agreement with intestinal-specific gene expression, highly expressed genes have enriched GATA-elements in their promoter regions and their functional properties are associated with processes that are characteristic for the intestine. We systematically mapped pre-mRNA cleavage and polyadenylation sites, or polyA sites, including more than 3000 sites that have previously not been identified. The detailed analysis of the 3'-ends of the nuclear mRNA revealed widespread alternative polyA site use (APA) in intestinally expressed genes. Importantly, we found that intestinal polyA sites that undergo APA tend to have U-rich and/or A-rich upstream auxiliary elements that may contribute to the regulation of 3'-end formation in the intestine.

Nilsen TW et al. (1988) Worm Breeder's Gazette "trans-splicing in Ascaris."

Bennett KL, Faser C, Nilsen TW

[Worm Breeder's Gazette, 1988]

It has been reported that Ascaris RNAs contain a splice leader sequence similar to that of Caenorhabditis tesh et al., WBG 10-1:67). When we first sent Ascaris des RNA samples to the laboratory of D. Hirsh, we also tested them with an oligonucleotide corresponding to the C. elegans spliced leader (SL) and found, as his group did, that a similar splice leader is present in Ascaris. Recently one of us (T. N.) has shown that the same 22 nt leader is present in the parasitic nematode causing human lymphatic filariasis, Brugia malayi (P.N.A.S., in press). Because Ascaris offers potential advantages to do biochemistry not available in Brugia, we have recently collaborated in further characterizing trans-splicing in Ascaris and in cloning the Ascaris leader sequence. As in C. elegans, we have found by Northern blot analysis that a small (~100nt) polyA-RNA species is recognized by the spliced leader, and multiple mRNAs are detected when polyA+ RNAs are used. All RNAs tested by Northern analysis including early embryonic (<30 cell), larval, oocyte and gut were positive, implying, but certainly not proving, no tissue or stage-specificity. By Southern blot at least two enzymes, ScaI and HaeIII, cut genomic DNA to an ~1 kb repeat. The same pattern of digestion was seen using the 5S ribosomal gene probe, indicating the trans-spliced leader is located in the 5S repeat, as found in C. elegans and in Brugia. An Ascaris genomic library constructed in lambda EMBL4 (Bennett and Ward, Dev. Bio. (1986) 118:141) was screened with the 22 mer SL oligonucleotide and with an oligonucleotide from the Brugia 5S genes. Positive lambda clones which contained the 5S gene and the sequence leader have been plaque purified; one has been subcloned and sequenced in part. We have found the Ascaris 22nt sequence leader is exactly the same as the C. elegans and Brugia one, with the rest of the small RNA sequence divergent. To determine the percentage of messages that contain the spliced leader, we have used polyA+ RNA from a mixed C. elegans population and from various Ascaris tissues and developmental stages. With 0.5 g of C. elegans polyA+ RNA, the SL oligonucleotide hybridizes to 10.6% as much RNA as does a labeled oligo dT probe. In the same assay using Ascaris RNAs, only 1.1-2.0% of the messages appear to contain the spliced leader in gut, oocyte and larval RNAs. While this one experiment implies that fewer messages are trans- spliced in Ascaris, the results could also be due to Ascaris messages having much longer polyA+ tails than C. elegans. However, our Northern blots also imply fewer Ascaris messages are trans-spliced when Ascaris and C. elegans polyA+ RNAs are compared.

FU, Lan et al. (2011) International Worm Meeting "The identification of the functional components of the male-specific CEM neurons."

Chow, King L., CHAN, Gus C. M., Fu, Lan

[International Worm Meeting, 2011]

CEMs are the only four male-specific cephalic neurons located in the head region of Caenorhabditis elegans. They are required for sex pheromone perception possibly for sensory input (Chasnov et al., 2007). However, their specific role and molecular function in this process is largely unknown. In order to define the cellular identity of CEM and active elements conferring its function, we are analyzing the CEMs' transcriptome using Illumina sequencing technique. Although ced-4 mutant hermaphrodites have non-functional CEMs preserved, these animals are not able to respond to sex pheromone. Ectopic fem-3 expression in the nervous system could masculinize CEMs and significantly raised the sensitivity of ced-4 hermaphrodites to sex pheromone. Some cellular properties of CEM could also be partially restored with this fem-3 expression. PolyA RNA from the functional CEMs was obtained from such masculinized ced-4 mutant hermaphrodites and was compared with the polyA RNA from ced-4 mutant hermaphrodites with feminized CEMs. FLAG-tagged poly(A)-binding protein (PABP) was expressed under the CEM-specific pkd-2 promoter and the protein product crosslinked with the CEM-specific polyA RNA. The uniquely expressed or highly enriched RNA species in these functional CEMs are recovered. These unique components reflecting the molecular properties of CEM will be presented, where their potential involvement of the biological activity in the CEMs will be discussed. (The study is supported by Research Grants Council, Hong Kong.).

Mangone, Marco et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The landscape of C. elegans 3'UTRs."

Gutwein, Michelle, Mis, Emily, Zegar, Charles, Harkins, Tim, Kim, John, Suzuki, Yukata, Thierry-Mieg, Jean, Rajewsky, Nikolaus, Han, Ting, Mangone, Marco, Sugano, Sumio, Mackowiak, Sebastian, Buffard, Pascal, Thierry-Mieg, Danielle, Prasad Manoharan, Arun, Khivansara, Vishal, Piano, Fabio, Gunsalus, Kris C, Kohara, Yuji, Salehi-Ashtiani, Kourosh

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Three-prime untranslated regions (3UTRs) of metazoan mRNAs contain numerous regulatory elements, yet the structure and developmental impact of 3UTRs remain largely uncharacterized. By integrating data from all Caenorhabditis elegans (C. elegans) developmental stages obtained by polyA capture, 3RACE, full-length cDNAs, and RNAseq, we define ~26,000 distinct mRNA 3 UTRs for ~85% of the 18,328 experimentally supported protein coding genes and refine the annotation of ~30% of gene models. Alternative 3UTR isoforms display widespread differential expression during development. Surprisingly, no canonical or variant polyadenylation signal (PAS) sequence is detec ted for 13% of polyA sites, most frequently among shorter alternative isoforms. Comparing trans-spliced and non trans-spliced genes reveals a strong correlation between the processing of transcript 5 and 3 ends: trans-spliced mRNAs possess longer 3UTRs and a higher frequency of no PAS or variant PAS motifs. We also predict conserved isoform-specific microRNA (miRNA) binding sites and identify additional evolutionarily constrained sequence blocks that may mediate 3UTR regulation. Thus, our data reveal a rich complexity of 3UTRs genome-wide and throughout development in C. elegans.

Tom Blumenthal et al. (2001) International C. elegans Meeting "A Global Analysis of the Caenorhabditis elegans Operons"

Kyle Duke, Alessandro Guffanti, Danielle Thierry-Mieg, Chris Link, Tom Blumenthal, Jean Thierry-Mieg, Donald Evans, Stuart Kim, Daniel Lawson

[International C. elegans Meeting, 2001]

Unlike genomes of other animals, the genomes of Caenorhabditis elegans and its close relatives contain numerous polycistronic transcription units similar to bacterial operons. The pre-mRNAs from these operons are processed into monocistronic mRNAs by 3' end formation and a specialized form of trans-splicing using the spliced leader, SL2. In order to determine how widespread operons are in the C. elegans genome, we have probed microarrays containing 17,817 predicted and known genes with a probe specific for mRNAs containing the SL2 sequence along with a control probe to polyA+ RNA. There are 1215 genes that form a separate group with high SL2/polyA+ ratios at the 99.9% level of confidence, and 86% of these appear to be downstream genes in operons, based on their genomic arrangements. We integrated this list with a list of ~400 genes in known operons and/or with confirmed SL2-containing cDNA clones. Of the genes previously confirmed to be SL2 trans-spliced, 91% are contained on the list of operons based on the microarray experiment, and almost all are in downstream positions. We estimate the genome contains ~850 operons, of which we provide experimental support for 807. On average, the operons contain 2.6 genes, and the longest contain seven genes. At least 13% of C. elegans genes are transcribed polycistronically. Operons are relatively common on chromosomes I and III, but much rarer on V and especially X. The average spacing between the polyA site of the upstream gene and the trans-splice site of the downstream gene is 126 bp. Most of the operons uncovered in this analysis have not been reported previously, and we can cite numerous new examples of co-transcription of genes encoding apparently functionally related proteins. We suggest the possibility that close inspection of the operon list could reveal heretofore unknown protein relationships.

Gengyo-Ando K et al. (1986) Worm Breeder's Gazette "progress in sequencing of the unc-15 paramyosin gene."

Kagawa H, Gengyo-Ando K

[Worm Breeder's Gazette, 1986]

DNAfragments from -HK2 clone which contained paramyosin gene were processed and get results as follows. Nucleotide sequences of the HindIII 5kb were and HindIII 1.4kb fragments were nearly completed. There many ORFs with predicted-helix protein structure. Polarity of ORF was determined from the amino acid sequence predicted with the nucleotide sequence. Many fusion proteins(Miller et al, Pro. N. A. S.(1986) 83, 2305-2309) were isolated and were characterized by immunoassay. One of them which was corresponded to the 3'end part of the HindIII 5kb fragment crossreacted with a monoclonal antibody which only crossreacted with full sized paramyosin. Our results and the information from Stephan D. Rioux(personal communication and the worm gazette, Vol. 9, No.2, p39), Tc1 insertion site of TR605 munant located at the 3' region of the gene. There were three polyA site in the HindIII 1.4 kb fragment. Although we are not sure which site is a real polyA site the result suggests that Tc1 insertion into 3' noncording region might decrease the termination of the message. C. a fashionable organism to study in Japan. I do hope you could have a time to attend a workshop in Japan near future.

Mangone, Marco et al. (2009) International Worm Meeting "The landscape of C.elegans 3'UTRs."

Mangone, Marco, Rajewsky, Nikolaus, Thierry-Mieg, Jean, Kim, John, Piano, Fabio, Ting, Han, Vidal, Marc, Chen, Kevin, Khivansara, Vishal, Prasad Manoharan, Arun, Zegar, Charles, Mis, Emily, Chen, Wei, Gunsalus, Kristin, Attie, Oliver, Kohara, Yuji, Salehi-Ashtiani, Kourosh, MacMenamin, Philip, Thierry-Mieg, Danielle

[International Worm Meeting, 2009]

Three-prime untranslated regions (3''UTRs) contain sequence elements used by RNA-binding proteins and regulatory RNAs such as microRNAs (miRNAs) to influence mRNA stability, translation and localization. However, the annotation of these regions within transcripts is generally incomplete. In C.elegans, which has a well annotated genome, about half of the ~20,000 curated genes in WS190 are not annotated with any 3''UTR and less than 10% are annotated with multiple or alternative 3''UTR isoforms. As part of the modENCODE project, we have developed a pipeline targeting ~7,000 genes using 3'' RACE and found at least one 3''UTR for around 90% of this targeted set. To analyze these data we have used different sequencing technologies that resulted in the identification of multiple distinct 3''UTR isoforms for over half of the targeted genes. In addition to 3''RACE, we have developed a technique to capture polyA ends followed by deep sequencing. This polyA-capture approach has resulted in over 2,000,000 polyA tags that map to ~12,000 genes across all major developmental stages and males, with the majority of these sequences mapping to full-length 3''UTRs. The depth of these data shows the remarkable complexity in the distribution of polyadenylation events in vivo. We have also manually curated 3''UTR boundaries using all available cDNAs, derived mostly from the 200,000 staged ESTs, and obtained 3''UTRs boundaries for ~11,500 genes. For about half of these (~5,000 genes), the data connect 3''UTRs to specific transcripts with known trans-spliced leader sequences at the 5'' end. Combining the results of these analyses has increased our knowledge of 3''UTR structures significantly, identifying novel 3''UTR isoforms for about half of all C.elegans protein-coding genes.

Montgomery, Taiowa et al. (2021) International Worm Meeting "piRNAs prevent runaway amplification of siRNAs from ribosomal RNAs, histone mRNAs, and other coding gene mRNAs"

Montgomery, Brooke, Reed, Kailee, Montgomery, Taiowa, Vijayasarathy, Tarah

[International Worm Meeting, 2021]

Piwi-interacting RNAs (piRNAs) are a largely germline-specific class of small RNAs found in animals. Although piRNAs are best known for silencing transposons, they regulate many different biological processes. Here we identify a role for piRNAs in preventing runaway amplification of small interfering RNAs (siRNAs) from certain genes, including ribosomal RNAs (rRNAs) and histone mRNAs. In C. elegans, rRNAs and some histone mRNAs are heavily targeted by piRNAs, which facilitates their entry into an endogenous RNA interference (RNAi) pathway involving a class of siRNAs called 22G-RNAs. Under normal conditions, rRNAs and histone mRNAs produce relatively low levels of 22G-RNAs. But if piRNAs are lost, 22G-RNA production is highly elevated. We show that 22G-RNAs produced downstream of piRNAs likely function in a feed-forward amplification circuit. Thus, our results suggest that piRNAs facilitate low-level 22G-RNA production while simultaneously obstructing the 22G-RNA machinery to prevent runaway amplification from certain RNAs. Histone mRNAs and rRNAs are unique from other cellular RNAs in lacking polyA tails, which may promote feed-forward amplification of 22G-RNAs. In support of this, we show that the subset of histone mRNAs that contain polyA tails are largely resistant to silencing in piRNA mutants. Despite hyperproduction of 22G-RNAs in piRNA mutants, the effects on histone and rRNA expression are modest and may have a negligible impact on germline development.