Mechanisms of Nuclear RNA Quality Control
To ensure the fidelity of gene expression, cells have evolved RNA quality control pathways that selectively degrade misprocessed RNAs. We study a specific nuclear RNA quality control pathway that uses components of the polyadenylation machinery. The pathway involves binding by the nuclear poly(A) binding protein, PABPN1, poly(A) tail extension by the poly(A) polymerases PAPα and PAPγ and decay by the nuclear exosome. We are interested in pursuing the many questions that 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
Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic human virus that uses the host cell machinery to express genes from its nuclear dsDNA genome. Outside of the context of viral infection, many KSHV genes are poorly expressed because host RNA quality control factors target their transcripts for degradation. To combat these host nuclear RNA decay pathways during infection, KSHV produces the ORF57 (Mta) protein, a viral RNA-binding protein that is essential for viral replication. ORF57 binds viral RNAs and interacts with human mRNA export factors to stabilize KSHV transcripts. We are currently exploring how ORF57 protects viral RNAs from nuclear decay and whether host mRNA export factors are essential for the stability of human transcripts in the nucleus.
Transcription Elongation
We recently performed a genome-wide CRISPR screen using a KSHV reporter with the goal of identifying novel components of RNA quality control pathways. Instead, we discovered that the transcription factor PNUTS (PPP1R10) is a negative regulator of KSHV gene expression. While PNUTS function has largely been associated with transcription termination at the end of genes, our data suggest it negatively regulates a subset of viral genes within the gene body. Impressively, depletion of PNUTS in human cells leads to robust increases in KSHV replication after lytic reactivation in cultured cells. Thus, we proposed that PNUTS acts as an intrinsic barrier to KSHV replication. Our ongoing experiments investigate the molecular mechanisms and targets of PNUTS during KSHV infection.
Intron Retention
Recent studies have revealed that thousands of RNAs in human cells are subject to intron retention. Generally, intron retention involves the regulated splicing of a specific intron within a pre-mRNA. Under specific conditions, the efficiency of splicing of that intron is increased or decreased to modulate production of the mature mRNA. Despite widespread intron retention in human cells, the mechanisms that regulate gene expression by intron retention remain largely undefined. Our lab focuses on the regulation of two intron-retained transcripts: MAT2A and OGT. MAT2A encodes the only S-adenosylmethionine (SAM) synthetase expressed in most cells while OGT encodes the sole nucleocytoplasmic O-GlcNAc transferase.
MAT2A
SAM is the most widely used metabolite next to ATP and is involved in nearly every cellular process. As such, cells need to carefully maintain SAM levels. 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 limited amounts of the co-factor SAM. METTL16 then induces splicing of the otherwise retained last intron to promote more mRNA. Methylation of additional hairpins in the 3´ UTR control stability of the resulting MAT2A mRNA. We further showed that METTL16 is the U6 snRNA methyltransferase and are currently seeking to validate additional targets of METTL16. We continue to define the mechanisms of this SAM-sensing pathway and its role in SAM homeostasis.
OGT
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, we previously identified a cis-acting intronic splicing silencer (ISS) that is necessary for intron retention. We currently seek to identify trans-acting factors and probe the biological significance of this regulatory pathway.