Messenger RNAs and noncoding transcripts undergo tightly regulated processing steps that are essential for their expression. The overarching goal of the Conrad Lab is to elucidate the mechanisms involved in posttranscriptional regulation of gene expression in the human cell nucleus. We are interested in how nuclear RNA splicing, polyadenylation, export, modification, and decay are coordinated to ensure proper gene expression. In our studies, we use molecular and cell biology techniques, but we also focus heavily on RNA processing in nuclear viruses.
When a virus invades a host cell, it usurps critical components of the cellular machinery to ensure its propagation and potentially causes disease. Therefore, an understanding of the interactions between a virus and its host cell is necessary to develop strategies to combat viral disease. Yet this is not the only reason to study virology. The history of molecular biology demonstrates that we gain considerable insight into cellular gene function by examining virus-host interactions. Thus, by studying viral factors we learn about essential mechanisms of human pathogens and we gain insights into the basic biology of human cells.
Our Research Projects
Our lab focuses on two KSHV lytic factors, the ORF57 protein, and PAN RNA. ORF57 (also called Mta) is essential for viral replication and all herpesviruses contain an ORF57 homolog.
Work from several labs has implicated ORF57 or its homologs in nearly every aspect of viral gene expression including transcription, mRNA export, 3´-end formation, viral pre-mRNA splicing, and translation. Our work focuses on the nuclear functions of ORF57 and our recently published work supports a role for ORF57 in polyadenylated nuclear RNA stability. These many reported activities raise an interesting question: How does a single viral protein achieve all of these functions?
In one model, ORF57 independently acts in each of these phases of gene expression. Alternatively, ORF57 may be limited to one or two “core” functions that lead to indirect effects in other processes in gene expression. For example, increased 3´end processing efficiency may lead to enhanced RNA stability. Our lab is interested in the mechanistic dissection of ORF57 activities to determine how this protein contributes to KSHV lytic infection.
Another factor we study in our lab is the polyadenylated nuclear RNA (PAN RNA; also known as nut-1 and T1.1), the most abundant KSHV lytic transcript. As much as 70% of the polyadenylated transcripts in a lytically infected cell are PAN RNA! PAN is a non-coding RNA of unknown function that is transcribed by RNA polymerase II, has a 3´ poly(A) tail, but is not exported from the nucleus.
The impressive nuclear abundance of PAN RNA is due to its high levels of transcription and to an RNA element, called the ENE, which increases transcript stability by interacting in cis with PAN RNA’s poly(A) tail. Our studies use this unique RNA to uncover the mechanisms and regulation of polyadenylated RNA stability in the mammalian cell nucleus.
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
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 studies, 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.
Intron Retention
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.
MAT2A
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 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 of this SAM-sensing pathway.
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, 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.
