Nuclear RNA processing and surveillance
Our aim is to understand the nuclear pathways that process newly transcribed RNAs and assemble RNA-protein complexes, the mechanisms that regulate these pathways and the surveillance activities that monitor their fidelity. Our research combines in vivo UV crosslinking and high-throughput sequencing to precisely identify sites of RNA-protein interaction and RNA-RNA basepairing, with genetics, biochemistry, transcriptomics and bioinformatics.
Applying a UV crosslinking approach (CRAC) to the RNA polymerases allowed high- resolution, strand-specific mapping of transcriptionally engaged RNA polymerase I, II and III. In each case, this revealed markedly uneven crosslinking efficiency along genes. We believe this reflects strong effects of chromatin structure on polymerase processivity at a locallevel. In the case of RNAPII, we further mapped the distribution of multiple modified forms of the polymerase (mCRAC) and applied a Hidden Markoff Model (HMM) to help interpret the resulting, highly complex datasets (Milligan et al. 2016). Analyses of RNA polymerase III showed that many tRNA genes generate long, 3´-extended forms due to read-through of the canonical poly(U) terminators (Turowski et al. 2016). The steady-state levels of 3´-extended pre-tRNA transcripts are low, due to targeting by the nuclear surveillance machinery (Figure 1). In addition, several previously unidentified RNA polymerase III transcripts were mapped.
Long-standing observations by the group indicated that the activity of nuclear RNA degradation by the exosome nuclease complex is responsive to nutrient availability. Our recent work revealed that alterations in nuclear surveillance contribute, both positively and negatively, to the rapid remodeling of gene expression following nutrient shift (Figure 2). This identified nuclear RNA surveillance as an actively regulated step in gene expression (Bresson et al. 2017). It seems likely that changes in nuclear RNA degradation pathways will play important roles in other situations that require large scale reprogramming of gene expression, such as developmental steps in metazoans.
A major function of chromatin is the regulation of gene expression in the form of RNA transcripts. We therefore predicted extensive interplay between the transcriptome and chromatin-associated factors. Two prominent chromatin modifications are the methylation of histone H3 at lysine 4 (H3K4) and lysine 36 (H3K36), catalyzed by the Set1 and Set2 methyltransferases, respectively. We have recently shown that both Set1 and Set2 bind RNA in vivo. Set1 was strongly enriched on non-coding RNAs that show Set1-dependent transcriptional silencing. Interactions between Set1 and RNA are predominately mediated by RRM2 and deletion of this region reduced the chromatin association of Set1, accompanied by reduced levels of H3K4 tri-methylation and increased di-methylation.
Together, these findings increased our understanding of the interdependency of RNA synthesis and surveillance pathways.
Milligan, L., Huynh-Thu, V.A., Delan-Forino, C., Tuck, A., Petfalski, E., Lombraña, R., Sanguinetti, G., Kudla, G. and Tollervey, D. (2016) Strand- specific, high-resolution mapping of modified RNA polymerase II. Mol. Syst. Biol.,12, 874. doi: 10.15252/msb.20166869. PMID: 27250689
Turowski, T.W., Lesniewska, E., Delan-Forino, C., Sayou, C., Boguta, M., Tollervey, D. (2016) Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts. Genome Res., 26, 933-944. PMID: 27206856
Bresson, S., Tuck, A., Staneva, D. and Tollervey, D. (2017) Nuclear RNA decay pathways aid rapid remodeling of gene expression in yeast. Mol. Cell, 65, 787-800. PMID: 28190770