Biology subjects

Brow, D. A.

Publications and source records attributed to Brow, D. A..

4 recordsLinked to original sources

bioRxiv2025

Direct RNA-seq provides evidence for an antiterminator function of the yeast Hrp1 protein on RNA polymerase II transcripts

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.

molecular biology↗

bioRxiv2025

Substitutions in RNA-binding protein Hrp1 map a potential interaction surface with the yeast RNA polymerase II elongation complex

Anti-termination 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 anti-termination 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 and cold-sensitive growth, as does Asp but not 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 anti-termination 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. A targeted selection for suppressors of Rpb3-K9E in HRP1 yielded substitutions in RRMs 1 and 2, an essential Met- and Gln-rich region C-terminal to the RRMs, and other 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 terminator readthrough. The Rpb3-K9E-suppressor substitutions in Hrp1 likely weaken binding to the RNAP II elongation complex, compensating for Rpb3-K9E.

molecular biology↗

bioRxiv2022

Yeast U6 snRNA made by RNA polymerase II is less stable but functional

U6 small nuclear (sn)RNA is the shortest and most conserved snRNA in the spliceosome and forms a substantial portion of its active site. Unlike the other four spliceosomal snRNAs, which are synthesized by RNA polymerase (RNAP) II, U6 is made by RNAP III. To determine if some aspect of U6 function is incompatible with synthesis by RNAP II, we created a U6 snRNA gene with RNAP II promoter and terminator sequences. This "U6-II" gene is functional as the sole source of U6 snRNA in yeast, but its transcript is much less stable than U6 snRNA made by RNAP III. Addition of the U4 snRNA Sm protein binding site to U6-II increased its stability and led to formation of U6-II*Sm complexes. We conclude that synthesis of U6 snRNA by RNAP III is not required for its function and that U6 snRNPs containing the Sm complex can form in vivo. The ability to synthesize U6 snRNA with RNAP II relaxes sequence restraints imposed by intragenic RNAP III promoter and terminator elements and allows facile control of U6 levels via regulators of RNAP II transcription.

molecular biology↗

bioRxiv2020

Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes

Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2',3' cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1-7 loads onto RNA from the 3' end and removal of the Lsm1 C-terminal region allows Lsm1-7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.

biochemistry↗