New Roles in RNA Stability and Transcription for the RNA Export Protein, REF/Aly
Stubbs, Sarah Elizabeth Hulsey
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Pre-mRNA processing consists of a series of events including capping, splicing, and polyadenylation, which occur co-transcriptionally, are interdependent, and are coupled to other steps in gene expression including transcription and RNA export. For example, in mammalian cells the TREX (transcription-export) complex is deposited on an mRNA in a splicing-dependent fashion, leading to more efficient mRNA export. In yeast, TREX is additionally implicated in RNA quality control pathways. Using factors derived from the Kaposi’s sarcoma-associated herpesvirus (KSHV), we established a role for the TREX component, REF/Aly, in nuclear RNA stability. We found that REF/Aly is recruited to the KSHV nuclear noncoding PAN RNA by the viral protein, ORF57, and this recruitment correlates with ORF57-mediated stabilization of PAN RNA. Additionally, REF/Aly is sufficient to increase the nuclear abundance and half-life of PAN RNA, but is not sufficient to promote its export. REF/Aly appears to protect the poly(A) tail from deadenylation and REF/Aly-stabilized transcripts are further adenylated over time, consistent with previous reports linking poly(A) tail length with nuclear RNA surveillance and supporting a broader conservation of RNA quality control mechanisms from yeast to human. Pre-mRNA processing is also mechanistically coupled to transcription with RNA pol II serving as a platform to recruit RNA processing factors to nascent transcripts. Using an RNA-Seq approach, we suggest a role for REF/Aly in transcription in addition to its well-defined role in RNA export. RNA-Seq analysis identified a subset of transcripts with decreased expression in both nuclear and cytoplasmic fractions upon REF/Aly knockdown. Interestingly, we find that REF/Aly does not affect the stability of candidate transcripts, however, REF/Aly depletion affects pol II density on target genes, implying a role for REF/Aly in pol II recruitment. Furthermore, REF/Aly binds directly to candidate transcripts, supporting a direct effect of REF/Aly on candidate gene transcription. Our data are consistent with the model that REF/Aly is involved in linking splicing with transcription efficiency. Taken together, our data suggest that the importance of REF/Aly is not limited to RNA export, but that REF/Aly is also critical for gene expression at the level of RNA stability and transcription.