Abstract
Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3’ end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3’ end formation. Tiling arrays and RNAPII mapping data revealed 3’ extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression.
Highlights
Budding yeast Npl3 comprises two RNA binding domains (RBDs) and a C-terminal domain that is rich is Arg, Gly, Ser and Tyr residues
We found that Npl3 binds diverse sites on large numbers of transcripts, and that the loss of Npl3 results in transcriptional readthrough on many genes
This underlines the importance of faithful termination for the correct regulation of gene expression
Summary
Budding yeast Npl comprises two RNA binding domains (RBDs) and a C-terminal domain that is rich is Arg, Gly, Ser and Tyr residues This structure shows similarities to the SR (SerArg rich) class of metazoan pre-mRNA binding proteins [1,2]. In the ‘torpedo’ pathway, the nascent RNA molecule is cleaved at the polyA site and the released 3’ fragment of the transcript still bound by RNAPII is degraded by the 5’-3’ exonuclease Rat. In the ‘torpedo’ pathway, the nascent RNA molecule is cleaved at the polyA site and the released 3’ fragment of the transcript still bound by RNAPII is degraded by the 5’-3’ exonuclease Rat1 This is proposed to destabilize the polymerase complex. Analyses on reporter constructs indicated that Npl can act as an anti-terminator, by antagonizing cleavage factor 1 (CF1) binding and restricting the use of cryptic poly (A) sites [4,7,11]
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