Antitermination factors for eukaryotic RNA polymerase II (RNAP II) that are released upon binding sequences in the terminator of nascent transcripts were proposed almost 40 years ago but few candidates have been found. Here we report genetic evidence that the yeast nuclear RNA-binding protein Hrp1, also known as Nab4 and CF1B, acts as an RNAP II antitermination factor. A Lys to Glu substitution at residue 9 (K9E) of the Rpb3 subunit of RNAP II causes readthrough of Nrd1-Nab3-Sen1-dependent (NNS) terminators in a reporter gene and cold-sensitive growth, as does an Asp but not an Ala, Met, Arg, or Gln substitution. These allele-specific phenotypes and the location of Rpb3-K9 suggests substitution with Glu or Asp stabilizes binding of an antitermination factor via a salt bridge. A genome-wide selection for suppressors of the cold-sensitivity of Rpb3-K9E yielded an Arg to Gly substitution at residue 317 of Hrp1 in RNA recognition motif 2 (RRM2), consistent with the hypothesis. Nanopore direct RNA-seq revealed strong readthrough of endogenous NNS terminators due to Rpb3-K9E and confirmed their partial suppression by Hrp1-R317G. A targeted selection for suppressors of Rpb3-K9E in HRP1 yielded substitutions in RRMs 1 and 2 and in an essential Met- and Gln-rich low complexity domain, as well as early nonsense mutations. We propose that Hrp1 binds to the RNAP II elongation complex via these regions to promote elongation and, in the presence of Rpb3-K9E, is less rapidly released upon binding terminator sequences in the nascent transcript, resulting in readthrough. The Rpb3-K9E-suppressor substitutions in Hrp1 are proposed to weaken binding to the RNAP II elongation complex, compensating for Rpb3-K9E.

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Journal Article

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Wang M, Arora P, Kaplan CD, Brow DA

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