Caenorhabditis elegans mutants deleted for TDP-1, an ortholog of the neurodegeneration-associated RNA-binding protein TDP-43, display only mild phenotypes. Nevertheless, transcriptome sequencing revealed that many RNAs were altered in accumulation and/or processing in the mutant. Analysis of these transcriptional abnormalities demonstrates that a primary function of TDP-1 is to limit formation or stability of double-stranded RNA. Specifically, we found that deletion of tdp-1: (1) preferentially alters the accumulation of RNAs with inherent double-stranded structure (dsRNA); (2) increases the accumulation of nuclear dsRNA foci; (3) enhances the frequency of adenosine-to-inosine RNA editing; and (4) dramatically increases the amount of transcripts immunoprecipitable with a dsRNA-specific antibody, including intronic sequences, RNAs with antisense overlap to another transcript, and transposons. We also show that TDP-43 knockdown in human cells results in accumulation of dsRNA, indicating that suppression of dsRNA is a conserved function of TDP-43 in mammals. Altered accumulation of structured RNA may account for some of the previously described molecular phenotypes (e.g., altered splicing) resulting from reduction of TDP-43 function.

Nearly 15% of the ~20,000 C. elegans genes are contained in operons, multigene clusters controlled by a single promoter. The vast majority of these are of a type where the genes in the cluster are ~100 bp apart and the pre-mRNA is processed by 3' end formation accompanied by trans-splicing. A spliced leader, SL2, is specialized for operon processing. Here we summarize current knowledge on several variations on this theme including: (1) hybrid operons, which have additional promoters between genes; (2) operons with exceptionally long (> 1 kb) intercistronic regions; (3) operons with a second 3' end formation site close to the trans-splice site; (4) alternative operons, in which the exons are sometimes spliced as a single gene and sometimes as two genes; (5) SL1-type operons, which use SL1 instead of SL2 to trans-splice and in which there is no intercistronic space; (6) operons that make dicistronic mRNAs; and (7) non-operon gene clusters, in which either two genes use a single exon as the 3' end of one and the 5' end of the next, or the 3' UTR of one gene serves as the outron of the next. Each of these variations is relatively infrequent, but together they show a remarkable variety of tight-linkage gene arrangements in the C. elegans genome.

TDP-1 is the Caenorhabditis elegans ortholog of mammalian TDP-43, which is strongly implicated in the etiology of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We discovered that deletion of the tdp-1 gene results in enhanced nuclear RNA interference (RNAi).

The heptad repeat of the RNA polymerase II (RNAPII) C-terminal domain is phosphorylated at serine 5 near gene 5 ends and serine 2 near 3 ends in order to recruit pre-mRNA processing factors. Ser-5(P) is associated with gene 5 ends to recruit capping enzymes, whereas Ser-2(P) is associated with gene 3 ends to recruit cleavage and polyadenylation factors. In the gene clusters called operons in Caenorhabditis elegans, there is generally only a single promoter, but each gene in the operon forms a 3 end by the usual mechanism. Although downstream operon genes have 5 ends, they receive their caps by trans splicing rather than by capping enzymes. Thus, they are predicted to not need Ser-5 phosphorylation. Here we show by RNAPII chromatin immunoprecipitation (ChIP) that internal operon gene 5 ends do indeed lack Ser-5(P) peaks. In contrast, Ser-2(P) peaks occur at each mRNA 3 end, where the 3-end formation machinery binds. These results provide additional support for the idea that the serine phosphorylation of the C-terminal domain (CTD) serves to bring RNA-processing enzymes to the transcription complex. Furthermore, these results provide a novel demonstration that genes in operons are cotranscribed from a single upstream promoter.Pre-mRNAs of protein-coding genes must be processed into mature mRNAs for translation. This transcription is carried out by RNA polymerase II (RNAPII) in association with a wide range of nuclear proteins that serve at different stages in the transcription cycle. Shortly after the nascent RNA emerges from RNAPII, its 5Ј end is cotranscriptionally capped (6,8,26,29). The addition of the cap is performed in three steps: first, the 5Ј phosphate is removed by RNA triphosphatase; second, GMP is added by RNA guanyltransferase; and third, the cap is methylated by RNA methyltransferase (33). At the other end of the gene, the pre-mRNA is cotranscriptionally cleaved by the 3Ј-end formation machinery composed of the multisubunit proteins cleavage and polyadenylation specificity factor (CPSF) and cleavage-stimulatory factor (CstF) as well as several additional proteins. However, transcription does not terminate (i.e., release from the template) until the polymerase has continued synthesizing RNA for an additional kilobase or more (10,12,15). The 3Ј-end formation machinery, and perhaps pre-mRNA cleavage itself, plays a key role in the termination event. One popular idea is that cleavage exposes a free 5Ј phosphate end on the downstream RNA, thereby allowing access to the 5Ј-to-3Ј exonuclease XRN2 (11,18,38,39).To accommodate the large number of proteins required for these and other cotranscriptional events, RNAPII includes a unique and flexible tail-like domain at the carboxy terminus of its largest subunit, referred to as its carboxy-terminal domain (CTD). The CTD is composed of numerous heptad repeats with the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , a sequence conserved among all eukaryotes (35). The number of these heptad repeats correlates with genomic complexity, varying from 26 repeats in th...

Transcription termination is mechanistically coupled to pre-mRNA 3 0 end formation to prevent transcription much beyond the gene 3 0 end. C. elegans, however, engages in polycistronic transcription of operons in which 3 0 end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3 0 ends. These 3 0 peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3 0 end formation factor CstF. This pattern occurs at all 3 0 ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3 0 end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3 0 ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3 0 cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3 0 end machinery to the transcription complex.