INVESTIGATING THE BASES OF THE SPECIFICITY OF THE RESTRICTOR COMPLEX FOR THE TERMINATION OF NONCODING TRANSCRIPTION

Rampant transcription initiation occurring at the tens of thousands of cis-regulatory regions in the genome can be detrimental to the normal functioning of the cell. Several transcription termination mechanisms act to keep the initiation of non-productive transcription in check. The Restrictor complex, composed of the adaptor protein WDR82 and the RNA-binding protein ZC3H4, suppresses processive transcription of thousands of noncoding RNAs (ncRNA) in mammalian genomes. The mechanism of Restrictor-mediated termination is not known but does not involve cleavage of the nascent RNA, setting it apart from known mammalian termination machineries. Furthermore, it is uniquely specific for noncoding transcription. Here, we show that ZC3H4 binds its non-coding RNA targets with high specificity, thereby bringing about Restrictor termination activity. We show that ZC3H4 RNA binding occurs at the very 5’ end of the transcript, to a specific RNA consensus sequence. We found that the activity of the Restrictor complex is more widespread than previously thought, with its depletion resulting in processive transcription at a vast number of previously undetectable ncRNAs. Further, we show that PNUTS, a protein implicated in transcription termination and also in the Restrictor-mediated pathway, directly binds to nascent RNA originating from different genomic loci where PNUTS’ phosphatase binding partner PP1 is known to act. We show that among the PNUTS-bound RNAs are Restrictor-sensitive ncRNAs, thereby providing a basis for the Restrictor-PNUTS synergy recently reported. Finally, I show evidence of sequence specificity of PNUTS’ RNA binding, suggesting that the underlying RNA sequence facilitates recruitment of PNUTS-PP1 for targeted dephosphorylation events.