Abstract
Hrp1/Nab4 is an essential nuclear RNA-binding protein that was first identified in the yeast Saccharomyces cerevisiae as a cleavage and polyadenylation factor for mRNAs, CF1B, but was later shown to promote termination of some short, noncoding transcripts via the "NNS" termination pathway. Hrp1 binds (UA)(n) repeats found in both mRNA 3'-UTRs and short noncoding RNA terminators, but its function in 3'-end formation is not fully understood. Our past microarray transcriptome analysis of the heat-sensitive hrp1-7 allele suggested Hrp1 functions in antitermination of RNA polymerase II (RNAP II) on protein-coding genes. The hrp1-7 allele has four substitutions and one, M191T, was shown to be primarily responsible for NNS terminator readthrough. Here we show that Hrp1-7 protein has a four-fold and Hrp1-M191T a two-fold decreased affinity for (UA)(5) in vitro. We used nanopore direct RNA sequencing to assess the transcriptome-wide effects of hrp1-7 and hrp1-M191T, which identified new examples of Hrp1-dependent small nucleolar RNA terminators and mRNA attenuators (regulatory terminators in the 5'-UTR and ORF). For some genes, including HRP1, attenuated transcript reads outnumber the corresponding mRNA reads in exponentially growing wild-type cells. We also observed widespread changes in mRNA polyA site selection. In the hrp1-M191T strain the polyA site shifts are mostly downstream, while in the hrp1-7 strain upstream and downstream shifts are more similar in frequency. Our results are consistent with a model in which some of the four substitutions in hrp1-7 weaken the affinity of Hrp1 for the RNAP II elongation complex, while others interfere with its recognition of specific sequences in nascent transcripts that would normally promote its release from the elongation complex.