Genes involved in pre-mRNA 3′-end formation and transcription termination revealed by a lin-15 operon Muv suppressor screen

Abstract

RNA polymerase II (Pol II) transcription termination involves two linked processes: mRNA 3-end formation and release of Pol II from DNA. Signals for 3 processing are recognized by a protein complex that includes cleavage polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF). Here we identify suppressors encoding proteins that play roles in processes at the 3 ends of genes by exploiting a mutation in which the 3 end of another gene is transposed into the first gene of the Caenorhabditis elegans lin-15 operon. As expected, genes encoding CPSF and CstF were identified in the screen. We also report three suppressors encoding proteins containing a domain that interacts with the C-terminal domain of Pol II (CID). We show that two of the CID proteins are needed for efficient 3 cleavage and thus may connect transcription termination with RNA cleavage. Furthermore, our results implicate a serine/arginine-rich (SR) protein, SRp20, in events following 3-end cleavage, leading to termination of transcription.Caenorhabditis elegans operon ͉ RNA polymerase II C-terminal domain ͉ SRp20 ͉ CTD T ermination of RNA polymerase II (Pol II)-mediated transcription plays an important role in gene regulation and involves two linked processes: 3Ј-end formation and release of the Pol II from the DNA (1). Accordingly, termination of Pol II requires both the presence of an intact 3Ј-processing signal and several 3Ј-end processing factors including the cleavage and polyadenylation specificity factor (CPSF) and the cleavage stimulation factor (CstF) (2-6). Several of the polyadenylation factors are associated with the C-terminal domain (CTD) of the largest subunit of Pol II, which is known to be required for efficient 3Ј processing (7-10). Much is known about what is required for 3Ј-end processing, but which factors specifically act in transcription termination and how these factors cause the Pol II complex to terminate are not entirely clear. The known link between 3Ј-end formation and transcription termination has led to multiple models to explain transcription termination.Two such models have been proposed to explain the connection between 3Ј-end processing and transcription termination. One model, known as the ''allosteric'' model, proposes that termination is triggered by a conformational change of the Pol II complex that occurs on the emergence of the polyadenylation sequences (3). Another model, known as the ''torpedo'' model, proposes that termination is triggered subsequent to the cleavage event by the exonuclease XRN-2. When cleavage occurs at the poly(A) polymerase site, Pol II continues to transcribe a now-uncapped downstream RNA. This RNA is subject to degradation by the 5Ј-3Ј exonuclease XRN-2; according to the torpedo model, termination occurs when the exonuclease collides with Pol II (11-13). Recently, a unified allosteric-torpedo model has been proposed to explain new experimental evidence in support of both the earlier models (14,15).One commonality between the three models of termination is the obl...

Transcription termination by nuclear RNA polymerases

“…These experiments, together with the observation that knockdown of p54nrb interferes with Xrn2 recruitment and termination, suggest that Xrn2 is recruited to the cleavage/polyadenylation machinery via p54nrb/PSF. The involvement of p54nrb/PSF in termination was recently confirmed in a genome-wide RNAi screen in C. elegans (Cui et al 2008).…”

“…Rtt103 is a Ser2 phosphorylated CTD-binding protein that cross-links to the 39-ends of genes in ChIP experiments, and copurifies with Rat1 and other subunits of the cleavage/polyadenylation complex (Kim et al 2004b). The Caenorhabditis elegans homolog of Rtt103, CIDS-1, was recently identified in a genome-wide RNAi screen for factors involved in termination, and deletion of cids-1 resulted in reduced 39-end cleavage, supporting the connection between cleavage and termination (Cui et al 2008). Additionally, Rat1 recruitment to the 39-end of a protein-coding gene was reduced by Pcf11 inactivation.…”

“…An intriguing study in C. elegans showed that an SR protein, SRp20, is implicated in mRNA transcription termination. Cui et al (2008) propose that SRp20 functions either in degradation of the cleaved 39-product or release of RNAPII from the template. ChIP assays combined with high-density microarrays showed that the splicing factor polypyrimidine tract-binding protein (PTB) is found at the 39-ends of many genes and in some cases many kilobases past the mature 39-end of the mRNA (Swinburne et al 2006).…”

Localization of RNAPII and 3′ end formation factor CstF subunits onC. elegansgenes and operons

“…42,43 but it very likely functions in 3 0 end formation. 44 In order to determine if the recruitment of the CstF proteins occurs at sites of 3 0 end formation, we performed ChIP-seq experiments with antibodies prepared against the worm CstF64 and CstF50 subunits. A representative example (soc-2) shows that both CstF subunits are strongly enriched at the site of 3 0 end formation (Fig.…”

“…42,43 Importantly, several 3 0 end formation factors were identified in a C. elegans suppressor screen for genes that play roles in 3 0 end formation. 44 We were interested in the question of how, the linkage of 3 0 end formation to transcription termination is broken at internal 3 0 end formation sites in operons.…”