Transcriptome-wide binding sites for components of the Saccharomyces cerevisiae non-poly(A) termination pathway: Nrd1, Nab3, and Sen1.

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.

Yeast Nrd1, Nab3, and Sen1 transcriptome-wide binding maps suggest multiple roles in post-transcriptional RNA processing.

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.

Substrate specificity of SIRT1-catalyzed lysine Nepsilon-deacetylation reaction probed with the side chain modified Nepsilon-acetyl-lysine analogs.

Peptides containing L-N(epsilon)-acetyl-lysine (L-AcK) or its side chain modified analogs were prepared and assayed using SIRT1, the prototypical human silent information regulator 2 (Sir2) enzyme. While previous studies showed that the side chain acetyl group of L-AcK can be extended to bulkier acyl groups for Sir2 (including SIRT1)-catalyzed lysine N(epsilon)-deacylation reaction, our current study suggested that SIRT1-catalyzed deacetylation reaction had a very stringent requirement for the distance between the alpha-carbon and the side chain acetamido group, with that found in L-AcK being optimal. Moreover, our current study showed that SIRT1 catalyzed the stereospecific deacetylation of L-AcK versus its D-isomer. The results from our current study shall constitute another piece of important information to be considered when designing inhibitors for SIRT1 and Sir2 enzymes in general.

Differential binding modes of the bromodomains of CREB-binding protein (CBP) and p300 with acetylated MyoD.

The recruitment of the bromodomains of CREB-binding protein (CBP) and p300 by the acetylated myogenic transcription factor MyoD was previously shown to be critical for the enhanced MyoD transcriptional activity following acetylation at its Lys99 and Lys102 positions. However, the modes of binding interactions of the bromodomains of CBP and p300 with acetylated MyoD have not been well-characterized. In the current study, by employing a panel of MyoD peptides encompassing the 99 and 102 positions, we showed that Lys99 monoacetylation and Lys99/Lys102 double acetylation defined the critical binding interfaces with the bromodomains of CBP and p300, respectively. This also represented the first identification of a recognition motif for the p300 bromodomain and revelation of the differential recognition motifs for the bromodomains of CBP and p300. This information could be exploited for developing novel tools for structural and functional studies of the highly homologous CBP and p300 transcriptional coactivators.

N(epsilon)-methanesulfonyl-lysine as a non-hydrolyzable functional surrogate for N(epsilon)-acetyl-lysine.

Through parallel studies on peptides containing N(epsilon)-methanesulfonyl-lysine or N(epsilon)-acetyl-lysine, N(epsilon)-methanesulfonyl-lysine as a replacement for N(epsilon)-acetyl-lysine was shown i) not to compromise the binding affinity for a bromodomain, ii) to confer resistance to human HDAC8 and SIRT1 (two distinct protein deacetylases), and iii) to confer only weak inhibition against human HDAC8 and SIRT1. These results suggested N(epsilon)-methanesulfonyl-lysine as a non-hydrolyzable functional surrogate for N(epsilon)-acetyl-lysine.