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PNUTS and CDK12 enhance KSHV gene expression independently of RNA quality control pathways
Author(s) -
Rosado-González Gabriela Tatiana,
Devlin Anne,
Conrad Nicholas
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.02714
Subject(s) - rna , biology , small interfering rna , gene knockdown , microbiology and biotechnology , rna binding protein , rna splicing , reporter gene , transfection , transcription (linguistics) , rna silencing , messenger rna , rna interference , gene , gene expression , genetics , linguistics , philosophy
Nuclear RNA quality control (QC) pathways degrade potentially deleterious transcripts arising from cryptic transcription. These QC pathways also target RNAs that are generated by improper mRNA capping, splicing, 3′ end formation or export. Kaposi’s sarcoma‐associated herpesvirus (KSHV) is a double‐stranded DNA virus whose transcripts avoid many of host RNA QC pathways, and it is used as a model to study mammalian cells RNA decay systems. One of the ways KSHV ensures this is by encoding ORF57 to protect viral transcripts from RNA decay systems. Previous pulse‐chase assay experiments with an unstable nuclear KSHV RNA demonstrated higher steady state KSHV RNA in presence of ORF57. Moreover, we recently found that the host proteins PPP1R10 (PNUTS) and CDK12 play important roles in controlling the steady‐state viral RNA levels. However, it was unknown if these processes work in the same pathway. The purpose of this study was to determine a possible synergistic or independent association between ORF57, PNUTS & CDK12 proteins in RNA stability. To accomplish this, PNUTS or CDK12 were knocked down with siRNAs in 293A‐TOA cells, followed by transient transfection of an ORF57‐responsive ORF59‐GFP reporter. The ORF59‐GFP reporter was co‐transfected with ORF57 or an empty vector control. We assayed RNA levels of the ORF59‐GFP reporter by northern blotting after PNUTS or CDK12 knockdown in the presence or absence of ORF57. Western blot verified that PNUTS and CDK12 were efficiently knocked down in cells treated with siRNA. Depletion of PNUTS or CDK12 resulted in higher steady‐state levels of ORF59 compared to controls. However, depletion of PNUTS or CDK12 in the presence of ORF57 resulted in higher steady‐state levels of ORF59. Our results suggest that PNUTS and CDK12 regulates RNA by a distinct mechanism than ORF57. Because ORF57 protects ORF59 from RNA QC pathways, we conclude that CDK12 and PNUTS do not affect RNA stability, but likely affect transcription or RNA processing.