Mechanisms of Nuclear RNA Quality Control
To ensure the fidelity of gene expression, cells have evolved RNA quality control pathways that selectively degrade RNAs that are misprocessed. We study a nuclear RNA degradation pathway that uses components of the polyadenylation machinery. The pathway involves binding by the nuclear poly(A) binding protein, PABPN1, hyperadenylation by the poly(A) polymerases PAPα/γ and decay by the nuclear exosome. Many questions remain about PABPN1-PAPα/γ-mediated RNA decay (PPD): How does PPD select its targets? What other factors are involved in PPD? Is PPD independent or redundant with other nuclear decay pathways? How do cells coordinate PPD with other steps in RNA biogenesis?
Viral Factors and Nuclear RNA Decay
The Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic human virus that expresses its genes using the host cell machinery. The KSHV protein ORF57 (Mta) is essential for viral replication and it protects viral RNAs from decay in the nucleus. We are currently testing whether ORF57 protects viral RNAs from PPD or other host cell RNA decay pathways. We also are exploring how ORF57 prevents nuclear RNA decay at the molecular level. In other studes, we are collaborating with Dr. Beatriz Fontoura’s lab to define interactions between influenza nuclear RNA, RNA export factors, and host nuclear quality control pathways.
Although thousands of mammalian RNAs are subject to intron retention and nuclear degradation, the processes that regulate gene expression by intron retention remain largely undefined. Our lab focuses on the regulation of two intron-retained transcripts that are subject to degradation by PPD: MAT2A and OGT. MAT2A encodes the only S-adenosylmethionine (SAM) synthetase expressed in most cells while OGT encodes the sole cellular O-GlcNAc transferase.
We defined a novel SAM feedback mechanism in which the m6A methyltransferase METTL16 controls the splicing of the MAT2A retained intron in response to SAM levels. Our working model proposes that under low SAM levels, METTL16 binds to a conserved hairpin (hp1) in the MAT2A 3´ UTR and remains bound presumably due to limiting amounts of the co-factor SAM. METTL16 then induces splicing of the otherwise retained last intron through its vertebrate conserved regions (VCRs). We also showed that METTL16 is the U6 snRNA methyltransferase and are currently seeking additional targets and functions of METTL16. Moreover, we continue to define the mechanisms this SAM-sensing pathway.
The OGT protein is responsible for nuclear and cytoplasmic O-GlcNAc addition to Ser/Thr, a common post-translational modification. Retention of the fourth intron of OGT RNA is regulated in response to cellular O-GlcNAc levels. Moreover, a cis-acting intronic splicing silencer (ISS) is necessary for intron retention. We currently seek to identify trans-acting factors and further probe the biological significance of this regulatory pathway.