RNA-protein crosslinking identifies novel targets for the nuclear RNA surveillance machinery
The RNA binding proteins Nrd1 and Nab3 function in transcription termination by RNA Pol II, acting via interactions with the CTD of the largest polymerase subunit, particularly on snRNA and snoRNA genes. They also participate in nuclear RNA surveillance and ncRNA degradation, functioning together with the exosome and the Trf-Air-Mtr4 polyadenylation (TRAMP) complexes. To better understand the signals for surveillance and ncRNA degradation, I applied an RNA-protein crosslinking approach in combination with Solexa sequencing. This approach identified in vivo binding sites for Nrd1, Nab3 and Trf4. Several million sequences were recovered and mapped to the yeast genome. This identified three classes of substrates: 1) Expected targets, including snRNAs, snoRNAs and characterized short ncRNAs. 2) Unknown but anticipated substrates, including several hundred previously uncharacterized ncRNAs that lie antisense to protein coding genes (asRNAs). 3) Unexpected targets, including many Pol III transcribed precursor RNAs. Bioinformatics analyses of the high-throughput sequencing data revealed that known binding motifs for Nrd1 and Nab3 were frequently recovered. Many recovered RNAs contained non-templated oligo(A) tails with an average of 2-5 nt length. This clearly distinguishes targets for surveillance machinery from polyadenylated mRNAs that get stabilized by polyadenylation (A70-90 in yeast). For a few selected, predicted asRNAs I was able to validate the crosslinking data by demonstrating that corresponding long RNAs are both detectable and increased by loss of Nrd1, Nab3, Trf4 or the exosome component Rrp6. Interestingly, loss of Nrd1 or Nab3 led to transcriptional read through on long asRNA transcripts. In addition, I have identified pre-TLC1 (telomerase RNA) as a target for the surveillance machinery. Processing of this long ncRNA was only poorly characterized in yeast but I could demonstrate that its transcription termination and maturation is mainly dependent on actions of the Nrd1-Nab3-Sen1, TRAMP4 and exosome complexes. It was previously reported that Nrd1-Nab3 acts only on short RNAs, due to the association with Ser5 phosphorylated CTD. My findings suggest that action of Nrd1- Nab3 is not exclusively on Ser5 phosphorylated form of the CTD. Unexpectedly the Pol II associated factors Nrd1 and Nab3 bound Pol III precursor transcripts. Surveillance of Pol III transcripts was dependent on Nrd1 and Nab3 since depletion of Nrd1 or Nab3 led to accumulation of pre-tRNAs. In addition, I could demonstrate that pre-RNase P RNA is oligoadenylated in vivo, which was dependent on Nrd1, Nab3 and Trf4. Together, my findings suggest a revised model of nuclear RNA surveillance in which Nrd1-Nab3 not only acts in co-transcriptional RNA recognition on Pol II transcripts but also post-transcriptionally on Pol III RNAs. The TRAMP complex is recruited to the defective RNA by the Nrd1-Nab3 complex, which remains associated with the RNA through the process of polyadenylation, until the exosome degrades the aberrant transcript.