Abstract
BackgroundNrd1 and Nab3 are essential sequence-specific yeast RNA binding proteins that function as a heterodimer in the processing and degradation of diverse classes of RNAs. These proteins also regulate several mRNA coding genes; however, it remains unclear exactly what percentage of the mRNA component of the transcriptome these proteins control. To address this question, we used the pyCRAC software package developed in our laboratory to analyze CRAC and PAR-CLIP data for Nrd1-Nab3-RNA interactions.ResultsWe generated high-resolution maps of Nrd1-Nab3-RNA interactions, from which we have uncovered hundreds of new Nrd1-Nab3 mRNA targets, representing between 20 and 30% of protein-coding transcripts. Although Nrd1 and Nab3 showed a preference for binding near 5′ ends of relatively short transcripts, they bound transcripts throughout coding sequences and 3′ UTRs. Moreover, our data for Nrd1-Nab3 binding to 3′ UTRs was consistent with a role for these proteins in the termination of transcription. Our data also support a tight integration of Nrd1-Nab3 with the nutrient response pathway. Finally, we provide experimental evidence for some of our predictions, using northern blot and RT-PCR assays.ConclusionsCollectively, our data support the notion that Nrd1 and Nab3 function is tightly integrated with the nutrient response and indicate a role for these proteins in the regulation of many mRNA coding genes. Further, we provide evidence to support the hypothesis that Nrd1-Nab3 represents a failsafe termination mechanism in instances of readthrough transcription.
Highlights
Nrd1 and Nab3 are essential sequence-specific yeast RNA binding proteins that function as a heterodimer in the processing and degradation of diverse classes of RNAs
Our data support the notion that Nrd1 and Nab3 function is tightly integrated with the nutrient response [30] and indicate a role for these proteins in the regulation of many mRNA coding genes
We have uncovered more than a thousand potential Nrd1Nab3 mRNA targets and our data indicate that Nrd1-Nab3 play an important role in the nutrient response and mitochondrial function
Summary
Nrd and Nab are essential sequence-specific yeast RNA binding proteins that function as a heterodimer in the processing and degradation of diverse classes of RNAs. These proteins regulate several mRNA coding genes; it remains unclear exactly what percentage of the mRNA component of the transcriptome these proteins control To address this question, we used the pyCRAC software package developed in our laboratory to analyze CRAC and PAR-CLIP data for Nrd1-Nab3-RNA interactions. To isolate direct protein-RNA interactions, cells are UV irradiated to forge covalent bonds between the protein of interest has markedly increased the sensitivity of the methodology and provided an unparalleled capability to identify protein-RNA interactions transcriptome-wide [3,5,7]. This approach is producing a lot of extremely valuable high-throughput sequencing data. We have recently developed a python package, dubbed pyCRAC, that conveniently combines many popular CLIP/CRAC analysis methods in an easy to use package
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