Genetic screen for suppression of transcriptional interference identifies a gain-of-function mutation in Pol2 termination factor Seb1.

Abstract

The system of long noncoding RNA (lncRNA)-mediated transcriptional interference that represses fission yeast phosphate homoeostasis gene <i>pho1</i> provides a sensitive readout of genetic influences on cotranscriptional 3'-processing and termination and a tool for discovery of regulators of this phase of the Pol2 transcription cycle. Here, we conducted a genetic screen for relief of transcriptional interference that unveiled a mechanism by which Pol2 termination is enhanced via a gain-of-function mutation, G476S, in the RNA-binding domain of an essential termination factor, Seb1. The genetic and physical evidence for gain-of-function is compelling: 1) <i>seb1-G476S</i> de-represses <i>pho1</i> and <i>tgp1</i>, both of which are subject to lncRNA-mediated transcriptional interference; 2) <i>seb1-G476S</i> elicits precocious lncRNA transcription termination in response to lncRNA 5'-proximal poly(A) signals; 3) <i>seb1-G476S</i> derepression of <i>pho1</i> is effaced by loss-of-function mutations in cleavage and polyadenylation factor (CPF) subunits and termination factor Rhn1; 4) synthetic lethality of <i>seb1-G476S</i> with <i>pho1</i> derepressive mutants <i>rpb1-CTD-S7A</i> and <i>aps1</i>∆ is rescued by CPF/Rhn1 loss-of-function alleles; and 5) <i>seb1-G476S</i> elicits an upstream shift in poly(A) site preference in several messenger RNA genes. A crystal structure of the Seb1-G476S RNA-binding domain indicates potential for gain of contacts from Ser476 to RNA nucleobases. To our knowledge, this is a unique instance of a gain-of-function phenotype in a eukaryal transcription termination protein.