RNA polymerase II synthesizes a diverse set of transcripts including both protein-coding and non-coding RNAs. One major difference between these two classes of transcripts is the mechanism of termination. Messenger RNA transcripts terminate downstream of the coding region in a process that is coupled to cleavage and polyadenylation reactions. Non-coding transcripts like Saccharomyces cerevisiae snoRNAs terminate in a process that requires the RNA–binding proteins Nrd1, Nab3, and Sen1. We report here the transcriptome-wide distribution of these termination factors. These data sets derived from in vivo protein–RNA cross-linking provide high-resolution definition of non-poly(A) terminators, identify novel genes regulated by attenuation of nascent transcripts close to the promoter, and demonstrate the widespread occurrence of Nrd1-bound 3′ antisense transcripts on genes that are poorly expressed. In addition, we show that Sen1 does not cross-link efficiently to many expected non-coding RNAs but does cross-link to the 3′ end of most pre–mRNA transcripts, suggesting an extensive role in mRNA 3′ end formation and/or termination.

RNA polymerase II transcribes both coding and noncoding genes, and termination of these different classes of transcripts is facilitated by different sets of termination factors. Pre-mRNAs are terminated through a process that is coupled to the cleavage/ polyadenylation machinery, and noncoding RNAs in the yeast Saccharomyces cerevisiae are terminated through a pathway directed by the RNA-binding proteins Nrd1, Nab3, and the RNA helicase Sen1. We have used an in vivo cross-linking approach to map the binding sites of components of the yeast non-poly(A) termination pathway. We show here that Nrd1, Nab3, and Sen1 bind to a number of noncoding RNAs in an unexpected manner. Sen1 shows a preference for H/ACA over box C/D snoRNAs. Nrd1, which binds to snoRNA terminators, also binds to the upstream region of some snoRNA transcripts and to snoRNAs embedded in introns. We present results showing that several RNAs, including the telomerase RNA TLC1, require Nrd1 for proper processing. Binding of Nrd1 to transcripts from tRNA genes is another unexpected observation. We also observe RNA polymerase II binding to transcripts from RNA polymerase III genes, indicating a possible role for the Nrd1 pathway in surveillance of transcripts synthesized by the wrong polymerase. The binding targets of Nrd1 pathway components change in the absence of glucose, with Nrd1 and Nab3 showing a preference for binding to sites in the mature snoRNA and tRNAs. This suggests a novel role for Nrd1 and Nab3 in destruction of ncRNAs in response to nutrient limitation.

The Saccharomyces cerevisiae Nrd1-Nab3 pathway directs the termination and processing of short RNA polymerase II transcripts. Despite the potential for Nrd1-Nab3 to affect the transcription of both coding and noncoding RNAs, little is known about how the Nrd1-Nab3 pathway interacts with other pathways in the cell. Here we present the results of a high-throughput synthetic lethality screen for genes that interact with NRD1 and show roles for Nrd1 in the regulation of mitochondrial abundance and cell size. We also provide genetic evidence of interactions between the Nrd1-Nab3 and Ras/protein kinase A (PKA) pathways. Whereas the Ras pathway promotes the transcription of genes involved in growth and glycolysis, the Nrd1-Nab3 pathway appears to have a novel role in the rapid suppression of some genes when cells are shifted to poor growth conditions. We report the identification of new mRNA targets of the Nrd1-Nab3 pathway that are rapidly repressed in response to glucose depletion. Glucose depletion also leads to the dephosphorylation of Nrd1 and the formation of novel nuclear speckles that contain Nrd1 and Nab3. Taken together, these results indicate a role for Nrd1-Nab3 in regulating the cellular response to nutrient availability.