Normal & Disease-Associated RNA Decay
The pioneer translation initiation complex is functionally distinct from but structurally overlaps with the steady-state translation initiation complex.
Half of the research in my lab focuses on NMD, which likely evolved to safeguard cells from potentially deleterious proteins produced as a consequence of routine mistakes in gene expression. In mammalian cells, these mistakes include inaccuracies in transcription initiation or pre-mRNA splicing, and ineffective somatic DNA rearrangements of the type that characterize the immunoglobulin and T-cell receptor genes. These mistakes often result in mRNAs having reading frames upstream of the usual reading frame, frameshift mutations that generate nonsense codons, or nonsense mutations. NMD also down-regulates a number of naturally occurring transcripts in mechanisms that maintain cellular homeostasis, including some selenoprotein mRNAs and alternatively spliced transcripts.
Recently, we tracked single mRNA molecules within cells in collaboration with Rob Singer's lab to verify that NMD indeed occurs on the cytoplasmic side of the nuclear envelope, with a half-life of less than one minute once the mRNA is in the cytoplasm. We have also characterized how newly synthesized mRNAs are remodeled during the pioneer round of translation and during the process of mRNA decay.
The other half of our research focuses on a mechanistically related pathway called Staufen1(STAU1)-mediated mRNA decay (SMD), or other pathways that are mediated by the double-stranded RNA-binding protein STAU1. SMD degrades mRNAs that harbor a STAU1-binding site in their 3'-untranslated regions (3'UTRs). Remarkably, depending on the particular mRNA, SBSs can be formed by (i) intramolecular base-pairing within a 3'UTR or (ii) intermolecular base-pairing between an mRNA 3'UTR and a long non-coding RNA (lncRNAs) or between an mRNA 3'UTR and another mRNA 3'UTR. Intermolecular base-pairing between an mRNA 3'UTR and a lncRNA or between two different mRNA 3'UTRs occurs via partially complementary Alu elements, thus describing new functions for these short interspersed elements (SINEs). We have shown the importance of SMD to myogenesis, keratinocyte motility, and the migration and invasion of human pancreatic adenocarcinoma cells.
Like microRNAs, more than one lncRNA or mRNA can target a single mRNA, and a single lncRNA can target more than one mRNA. Only those strands of a mRNA-lncRNA or mRNA-mRNA duplex that are translated are subject to SMD, explaining why we have yet to detect a lncRNA targeted for SMD. We have named these lncRNAs
½-sbsRNAs since they constitute one strand of an SBS. Notably, the STAU2 paralog of STAU1 also functions in SMD and, like STAU1, can homodimerize as well as form heterodimers with STAU1. In fact, homo- and hetero-multimers may exist. We have determined the X-ray crystal structure of a dimerization interface, which involves interactions between a new motif, which we have named the STAU-swapping motif, a degenerate RNA-binding domain. Remarkably, we have found that B SINES in mouse, which evolved independently of human Alu elements, also populate mRNA 3'UTRs and lncRNAs and can likewise base-pair to form SBSs and trigger SMD.
In other studies, we have found that STAU1 binding to 3'UTR inverted Alu elements competes with binding of the largely nuclear paraspeckle protein p54nrb and largely cytoplasmic protein kinase R (PKR) to mediate, respectively, the nuclear export and cytoplasmic translation of a number of mRNAs that contain these elements. Thus, STAU1 binding to 3'UTR inverted Alu elements, like removal of these elements by alternative RNA 3’-end cleavage and polyadenylation, obviates a PKR-mediated innate immune response to cytoplasmic 3'UTR inverted Alu elements.
Our on-going studies of NMD and dsRNA-binding protein-mediated RNA metabolism include computational analyses of cellular transcripts, proteomic studies of cellular RNA-binding proteins using mass spectrometry, and deep sequencing of cellular RNAs to gain insight into molecular mechanisms and how they can be attenuated for therapeutic purposes.
- Organizing principles of Mammalian nonsense-mediated mRNA decay. Annu Rev Genet. 47, 139-65. (2013 Nov 23).
- mRNA-mRNA duplexes that autoelicit Staufen1-mediated mRNA decay. Nat Struct Mol Biol. 20, 1214-20. (2013 Oct 01).
- STAU1 binding 3' UTR IRAlus complements nuclear retention to protect cells from PKR-mediated translational shutdown. Genes Dev. 27, 1495-510. (2013 Jul 01).