TERMINATION OF EXTRAGENIC TRANSCRIPTION BY THE RESTRICTOR COMPLEX: DISSECTING RNA BINDING SPECIFICITY

Controlling the precise spatiotemporal utilization of genomic information is crucial across all kingdoms of life. Dealing with the complexity of eukaryotic genomes is challenging due to the multitude of transcription initiation events generated by hundreds of thousands of cis-regulatory elements. Unchecked pervasive transcription can be costly, potentially compromising genome integrity by increasing the threats posed by transcription and disrupting the regulation of non-coding RNAs (ncRNAs). In this context, we recently identified the Restrictor complex, which plays a critical and non-redundant role in the termination of extragenic RNA Polymerase II (Pol II). The complex is composed of WDR82, an RNA Polymerase II-binding protein, and its RNA-binding partner ZC3H4. Restrictor predominantly terminates extragenic transcription, with limited impact on protein-coding genes. Numerous aspects of the functionality of the Restrictor complex remain enigmatic. My thesis work focused on the specific contribution of ZC3H4 to the activity of the complex. Through acute and rapid degron-mediated depletion of ZC3H4, I observed a significant upregulation of thousands of ncRNAs, many of which are polyadenylated and spliced. The extent of the upregulation was underestimated due to the unstable nature of Restrictor’s target RNAs, which normally are rapidly degraded. Analysis of the nascent transcriptome after DRB treatment and release revealed a greater number of targets regulated by ZC3H4, and that they are transcribed slower than protein-coding transcription units. Moreover, the genomic distribution of ZC3H4 revealed that it was recruited immediately downstream of transcription start sites, coinciding with the genomic distribution of initiating and early elongating Pol II, thus confirming that Restrictor acts in an early termination mechanism. In line with its reported RNA binding activity, the in-frame deletion of the putative RNA binding domain of ZC3H4 induced a partial upregulation of Restrictor targets. I further assessed its RNA binding activity both in vivo and in vitro, both of which yield a candidate binding motif for ZC3H4. This thesis highlights the role of sequence-specific recognition of nascent RNAs in the termination activity of the Restrictor complex.