Working in the Lab
Mechanism by Which Termination (Nonsense) Codons Elicit mRNA Decay
Posted on: Thursday, January 20, 2011.
Nonsense codons, caused by either frameshift or nonsense mutations, are responsible for an estimated one-third of inherited genetic diseases. We are particularly interested in understanding the changes in mRNP structure that occur during the pioneer round of translation and how the pioneer translation initiation complex is remodeled to the steady-state translation initiation complex.
NMD is a splicing-dependent and translation-dependent pathway that targets not only disease-associated but also naturally occurring transcripts (for recent review, see Maquat et al., 2010, Cell 142:368-74). Many of the naturally occurring transcripts are mistakes made during alternative splicing (Pan et al., 2006, Genes & Dev. 20:153-8). Currently, we are interested in further characterizing the pioneer round of translation, during which nonsense codon recognition leads to NMD (Ishigaki et al., 2001 Cell 106:607-617; Lejeune et al., 2002, EMBO J. 21:3536-3545; Woeller et al., 2008, EMBO Rep. 9:446-451). We have made important progress in identifying components of the pioneer translation initiation complex, which consists of the mostly nuclear but shuttling cap-binding proteins CBP80 and CBP20 at the mRNA cap, poly(A) binding proteins PABPN1 and PABPC1 at the mRNA poly(A) tail, and the exon junction complex (EJC) of proteins that includes the NMD factors UPF3 or UPF3X, UPF2, and, finally, UPF1 (Chiu et al., 2004, Genes & Dev. 18:645-754; Lejeune et al., 2004, Nat. Struct. Mol. Biol. 11:992-1000; Hosoda et al., 2006, Mol. Cell. Biol. 26:3085-3097). We have found that CBP80 promotes NMD by promoting the interaction between UPF1 and UPF2 (Hosoda et al., 2005, Nat. Struct. Mol. Biol. 12:893-901). More recent data indicate that CBP80 escorts UPF1 and its kinase SMG1 to join the translation termination complex eRF1-eRF3 at a premature termination codon and subsequently to join a downstream EJC (Hwang et al., 2010, Mol Cell 39:396-409). Joining results in UPF1 phosphorylation and, as a consequence, translational repression: phospho-UPF1 binds eIF3 of the 43S translation initiation complex that is poised at the initiation codon of an NMD target so as to inhibit 60S ribosomal subunit joining and, thus, formation of a translationally active 80S ribosome (Isken et al., 2008, 133:314-327). We have found that the pioneer round of translation promotes some but not all steps of mRNP remodeling to form the steady-state translation initiation complex. For example, the pioneer round of translation promotes EJC removal and the replacement of PABPN1 by PABPC1 but, remarkably, not the replacement of CBP80-CBP20 by eIF4E. Instead, the karyopherin importin-β mediates the replacement of cap-bound CBP80-CBP20 by eIF4E by interacting with importin-β, which is a stable constituent of cap-bound CBP80-CBP20 (Sato and Maquat, 2009, Gene & Dev. 23:2537-2550). Opportunities are available to research the degradative enzymology of NMD (Lejeune et al., 2003, Mol. Cell 12:675-687), factor function in NMD (Chiu et al., 2003, RNA 9:77-87; Brumbaugh et al., 2004, Mol. Cell 14:585-598; Matsuda et al., 2007, Nat. Struct. Mol. Biol. 14:974-979) and, in collaboration with Rob Singer (Albert Einstein College of Medicine), the spatial difference in cells between nucleus-associated and cytoplasmic NMD (Sato et al., 2008, Mol Cell 29:255-262).
Opportunities are also available to study a related mRNA decay pathway that we have named Staufen1(STAU1)-mediated mRNA decay (SMD) (Kim et al., 2005, Cell 120:195-208; Kim et al., 2007, EMBO J. 26:2670-2681). We have found that STAU1, which is a double-stranded RNA binding protein, recruits the NMD factor UPF1 to certain mRNA 3’-untranslated regions (3’UTRs) so as to elicit SMD in a translation-dependent fashion. Using microarray analyses, we have identified a number of mRNAs that are naturally down-regulated by SMD. Unlike NMD, SMD targets not only CBP80-CBP20-bound mRNA but also its remodeled product, eIF4E-bound mRNA. This makes sense for a conditionally regulated pathway. Remarkably, NMD and SMD are competitive pathways since UPF1 can bind either the UPF2 NMD factor or the STAU1 SMD factor but not both factors simultaneously; competition contributes to myogenesis and probably many other cellular processes (Gong et al., 20090 Genes & Dev. 23: 54-66). We are very excited about our finding that STAU1-binding sites (SBS) can be formed not only by intramolecular base-pairing in an mRNA 3’ UTR but also by intermolecular base-pairing between the Alu element of an mRNA 3’UTR and a partially complementary Alu element in one or more Alu element-containing long noncoding (lnc)RNAs (Gong and Maquat, 2011, Nature, in press). These lncRNAs are cytoplasmic and polyadenylated, and we refer to them as 1/2-sbsRNAs. Thus, we have defined unexpected roles for Alu elements and lncRNAs. Future studies aim to elucidate how mammalian cells utilize SMD to regulate gene expression. Included in these studies is identifying those dsRNA sequences in mRNAs that bind STAU1, defining STAU1-containing mRNA binding complexes, and characterizing the physiological significance of SMD. Additionally, we have evidence that STAU1 can affect mRNA metabolism independently of translation, and we are pursuing studies of this pathway.
Successful candidates will join a well-equipped group of interactive lab members with diverse backgrounds and broad expertise in newly remodeled labs. The University of Rochester is unique for its sizeable community of RNA researchers, its Center for RNA Biology: From Genome to Medicine, and its RNA Structure and Function Cluster, all of which include members of the Medical Center, in which the Maquat lab resides, as well as the College of Arts and Sciences across the street.
Interested individuals should send a C.V., including a description of past and on-going research, and the names and contact information of three references to Dr. Maquat
- Staufen-mediated mRNA decay. Wiley Interdiscip Rev RNA. In press. (2013 May 16).
- Control of myogenesis by rodent SINE-containing lncRNAs. Genes Dev. 27, 793-804. (2013 Apr 01).
- Staufen1 dimerizes through a conserved motif and a degenerate dsRNA-binding domain to promote mRNA decay. Nat Struct Mol Biol. 20, 515-24. (2013 Apr 01).