Post-transcriptional Gene Regulation by Double-stranded RNA-binding Proteins
Dr. Manuel Ares University of California Santa Cruz, Dr. Liran Carmel Hebrew University of Jerusalem, Dr. Clara Kielkopf, Dr. Bin Tian The Wistar Institute Cancer Center
Strategy to purify ½-sbsRNA1-containing RNP complexes.
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 mechanism (reviewed in Park and Maquat, 2013, Wiley Interdiscip Rev RNA 4:423-435). Using microarray analyses, we have identified a number of mRNAs that are naturally down-regulated by SMD.
Unlike NMD, SMD detectably targets not only CBP80-CBP20-bound mRNA but also its remodeled product, eIF4E-bound mRNA (Hosoda et al., 2005, Nat. Struct. Mol. Biol. 12:893-901). 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 undoubtedly many other cellular processes (Gong et al., 2009 Genes & Dev. 23: 54-66). Remarkably, STAU1-binding sites (SBS) can be formed not only by intramolecular base-pairing within an mRNA 3'UTR (Kim et al., 2005, Cell 120:195-208; Kim et al., 2007, EMBO J, 26:2670-2681) but also by intermolecular base-pairing either 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, 470:284-288) or between the 3'UTR Alu elements of two different mRNAs (Gong et al., 2013, Nat. Struct. Mol. Biol.)
The lncRNAs are cytoplasmic and polyadenylated, and we refer to them as ½-sbsRNAs. Thus, we have defined unexpected roles for Alu elements, lncRNAs and mRNAs. The STAU1 isoform, STAU2, also functions in SMD (Park et al., 2013, Proc. Nat. Acad. Sci. USA 110:405-412). Both STAU1 and STAU2 can homodimerize, and the two can also form heterodimers if not heteromultimers (Park et al., 2013, Proc. Nat. Acad. Sci. USA 110:405-412). Interactions involve a new motif that we call the STAU-swapping motif (SSM), and we have determined the X-ray crystal structure of the SSM of one molecule interacting with a degenerate dsRNA-binding domain of another molecule (Gleghorn et al., 2013 Nat. Struct. Mol. Biol. 20:515-524). We have also found that B SINES in mouse, which evolved independently of Alu elements in primates, also populate mRNA 3'UTRs and lncRNAs and can likewise base-pair to form SBSs and trigger SMD (Wang et al., 2013, Genes & Dev. 27:793-804).
STAU1 promotes 3'UTR IRAlus mRNAs export to the cytoplasm.
In other studies, we have found that STAU1 (and probably STAU2) 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. For reasons unknown, STAU1 binding to those 3'UTR inverted Alu elements that have been studied fails to trigger SMD.
STAU1 binding to 3'UTR IRAlus competes with p54nrb and
PRK binding to promote, respectively, mRNA export and translation.
Future studies aim to elucidate how mammalian cells utilize STAU and other dsRNA-binding proteins to regulate gene expression. Included in these studies is identifying those intramolecular and intermolecular sequences in 1/2-sbsRNAs and mRNAs that bind STAU, defining STAU-containing RNA-binding complexes, and characterizing the physiological significance of the various STAU-mediated pathways.
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