Fission yeast poly(A) polymerase active site mutation Y86D alleviates the rad24Δ asp1-H397A synthetic growth defect and up-regulates mRNAs targeted by MTREC and Mmi1.

TitleFission yeast poly(A) polymerase active site mutation Y86D alleviates the rad24Δ asp1-H397A synthetic growth defect and up-regulates mRNAs targeted by MTREC and Mmi1.
Publication TypeJournal Article
Year of Publication2023
AuthorsGarg A, Schwer B, Shuman S
JournalRNA
Volume29
Issue11
Pagination1738-1753
Date Published2023 Nov
ISSN1469-9001
KeywordsCatalytic Domain, mRNA Cleavage and Polyadenylation Factors, Mutation, RNA, Long Noncoding, RNA, Messenger, Schizosaccharomyces, Schizosaccharomyces pombe Proteins
Abstract

Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstream prt lncRNA that interferes with the pho1 mRNA promoter. lncRNA-mediated interference is alleviated by genetic perturbations that elicit precocious lncRNA 3'-processing and transcription termination, such as (i) the inositol pyrophosphate pyrophosphatase-defective asp1-H397A allele, which results in elevated levels of IP8, and (ii) absence of the 14-3-3 protein Rad24. Combining rad24Δ with asp1-H397A causes a severe synthetic growth defect. A forward genetic screen for SRA (uppressor of ad24 sp1-H397A) mutations identified a novel missense mutation (Tyr86Asp) of Pla1, the essential poly(A) polymerase subunit of the fission yeast cleavage and polyadenylation factor (CPF) complex. The pla1-Y86D allele was viable but slow-growing in an otherwise wild-type background. Tyr86 is a conserved active site constituent that contacts the RNA primer 3' nt and the incoming ATP. The Y86D mutation elicits a severe catalytic defect in RNA-primed poly(A) synthesis in vitro and in binding to an RNA primer. Yet, analyses of specific mRNAs indicate that poly(A) tails in pla1-Y86D cells are not different in size than those in wild-type cells, suggesting that other RNA interactors within CPF compensate for the defects of isolated Pla1-Y86D. Transcriptome profiling of pla1-Y86D cells revealed the accumulation of multiple RNAs that are normally rapidly degraded by the nuclear exosome under the direction of the MTREC complex, with which Pla1 associates. We suggest that Pla1-Y86D is deficient in the hyperadenylation of MTREC targets that precedes their decay by the exosome.

DOI10.1261/rna.079722.123
Alternate JournalRNA
PubMed ID37586723
PubMed Central IDPMC10578478
Grant ListP30 CA008748 / CA / NCI NIH HHS / United States
R01 GM134021 / GM / NIGMS NIH HHS / United States
R35 GM126945 / GM / NIGMS NIH HHS / United States

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