Multimerization of poly(rC) binding protein 2 is required for translation initiation mediated by a viral IRES

Bedard, Kristin; Walter, Brandon L.; Semler, Bert L.

doi:10.1261/rna.7070304

Cited by 47 publications

(40 citation statements)

References 32 publications

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“…PCBP2 was initially identified as a component of the a-complex of the human a-globin mRNA, which greatly increases mRNA stability (Kiledjian et al 1995). Currently, PCBP2 is best characterized as a facilitator of internal ribosomal entry site (IRES)-mediated translation of both viral and cellular transcripts (Parsley et al 1997;Evans et al 2003;Bedard et al 2004), but it is also implicated in other important biological processes, including transcriptional regulation and translational silencing (Makeyev and Liebhaber 2002). PCBP2 was previously described as a nuclear-cytoplasmic shuttling protein, which allows the protein to fulfill such diverse functions (Chkheidze and Liebhaber 2003).…”

Section: Introductionmentioning

confidence: 99%

Identification of PCBP2, a facilitator of IRES-mediated translation, as a novel constituent of stress granules and processing bodies

Fujimura¹,

Kano²,

Murata³

2008

RNA

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Recent advances in microscopic techniques have shed light on the roles of specific subcellular structures in the regulation of gene expression. One such structure is the stress granule (SG), which is engaged in stress-triggered translational arrest by sequestering pre-initiation complexes of translation. Recent studies revealed the spatial, compositional, and functional linkage of the SG to the processing body (P-body), another cytoplasmic structure that has been implicated in mRNA degradation and siRNA-or miRNA-mediated gene silencing. In this study, we report that PCBP2, a facilitator of IRES (Internal Ribosomal Entry Site)-mediated translation, is a novel constituent of the SG and P-body. Immunofluorescence studies revealed that while PCBP2 is diffusely distributed throughout the nucleoplasm and the cytoplasm, the protein is enriched in a subset of P-bodies under normal conditions. Upon exposure to heat and arsenic stress, PCBP2 became predominantly accumulated at the SG, but was still present in Dcp1a-positive P-bodies. Live-cell imaging revealed the dynamic association of PCBP2-enriched P-bodies and the SG, and FRAP experiments demonstrated that PCBP2 actively moves in and out of the SG and P-body. Taken together, these results suggest that PCBP2 shuttles between the cytoplasm and the two structures under stress. We propose that PCBP2 may be involved in stress-induced remodeling of mRNP complexes and that it may also play a role in the rapid transition of certain silenced mRNAs into a translationally active state. Additionally, given the property of PCBP2 as a nuclear-cytoplasmic shuttling protein, PCBP2 may play a role in directly targeting nascent mRNPs to specific P-bodies for storage.

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Section: Introductionmentioning

confidence: 99%

Identification of PCBP2, a facilitator of IRES-mediated translation, as a novel constituent of stress granules and processing bodies

Fujimura¹,

Kano²,

Murata³

2008

RNA

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“…The element in the viral RNA that constitutes the IRES adopts a complex RNA structure, likely stabilized by RNAbinding proteins (Belsham and Jackson 2000;Hellen and Sarnow 2001;Bedard et al 2004;Martínez-Salas and Fernán-dez-Miragall 2004). Experimental evidence for tertiary structure generated by RNA-RNA interactions is available for portions of IRES elements from FMDV (Ramos and Martí-nez-Salas 1999;Fernández-Miragall and Martínez-Salas 2003), HCV (Kieft et al 1999;Jubin et al 2000;Lyons et al 2001;Spahn et al 2001;Kieft et al 2002;Vos et al 2002;Lukavsky et al 2003;Rijnbrand et al 2004), and the intergenic region of insect picornavirus-like viruses (Jan and Sarnow 2002;Costantino and Kieft 2005).…”

Section: Introductionmentioning

confidence: 99%

Evidence of reciprocal tertiary interactions between conserved motifs involved in organizing RNA structure essential for internal initiation of translation

Fernández-Miragall¹,

Ramos²,

Ramajo³

et al. 2005

RNA

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Internal ribosome entry site (IRES) elements consist of highly structured RNA regions that determine internal initiation of translation. We have previously shown that the foot-and-mouth disease virus (FMDV) IRES contains a GNRA tetraloop spanning residues G 178 UAA 181 . Here we show that tertiary RNA interactions dependent on the GNRA motif determine the structural organization of the central domain. By using mutational analysis in combination with RNA probing, we have identified distant reciprocal interactions between the GNRA motif and the invariant region G 240 CACG 244 , termed motif A. Mutations in motif A caused a decrease in IRES activity as severe as the GUAG substitution in the GNRA motif. Substitutions in either GNRA or motif A sequences induced a common reorganization around the conserved R 199 AAA 202 stem-loop, suggesting that the latter contributes to stabilize the GNRA-motif A interaction. This finding was also consistent with a significant increase in the efficiency of RNA-RNA interactions determined in gel shift assays using as probe the hairpin that contains the GNRA motif compared to a transcript encompassing the entire apical region of the central domain. Thus, we propose that the central domain of the FMDV IRES contains a structural conformation essential for IRES activity stabilized by a tertiary contact involving residues in the GNRA tetraloop and motif A conserved sequences.

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“…This binding is mediated by KH1, which is the primary RNA-binding domain (62,69). A truncated form of PCBP2 missing the KH1 domain cannot rescue poliovirus translation in HeLa cell cytoplasmic extracts depleted of endogenous PCBP (6). The second domain (KH2) is responsible for multimerization of PCBP2, and this multimerization is required for RNA binding and translation initiation on the poliovirus IRES element (6).…”

mentioning

confidence: 99%

“…A truncated form of PCBP2 missing the KH1 domain cannot rescue poliovirus translation in HeLa cell cytoplasmic extracts depleted of endogenous PCBP (6). The second domain (KH2) is responsible for multimerization of PCBP2, and this multimerization is required for RNA binding and translation initiation on the poliovirus IRES element (6). Through mutational analysis, it was determined that the KH3 domain is also important for binding of PCBP2 to stem-loop IV (69).…”

mentioning

confidence: 99%

Cellular Protein Modification by Poliovirus: the Two Faces of Poly(rC)-Binding Protein

Perera

Daijogo

Walter

et al. 2007

J Virol

Self Cite

138

151

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During picornavirus infection, several cellular proteins are cleaved by virus-encoded proteinases. Such cleavage events are likely to be involved in the changing dynamics during the intracellular viral life cycle, from viral translation to host shutoff to RNA replication to virion assembly. For example, it has been proposed that there is an active switch from poliovirus translation to RNA replication mediated by changes in RNA-binding protein affinities. This switch could be a mechanism for controlling template selection for translation and negative-strand viral RNA synthesis, two processes that use the same positive-strand RNA as a template but proceed in opposing directions. The cellular protein poly(rC)-binding protein (PCBP) was identified as a primary candidate for regulating such a mechanism. Among the four different isoforms of PCBP in mammalian cells, PCBP2 is required for translation initiation on picornavirus genomes with type I internal ribosome entry site elements and also for RNA replication. Through its three K-homologous (KH) domains, PCPB2 forms functional protein-protein and RNA-protein complexes with components of the viral translation and replication machinery. We have found that the isoforms PCBP1 and -2 are cleaved during the mid-to-late phase of poliovirus infection. On the basis of in vitro cleavage assays, we determined that this cleavage event was mediated by the viral proteinases 3C/3CD. The primary cleavage occurs in the linker between the KH2 and KH3 domains, resulting in truncated PCBP2 lacking the KH3 domain. This cleaved protein, termed PCBP2-⌬KH3, is unable to function in translation but maintains its activity in viral RNA replication. We propose that through the loss of the KH3 domain, and therefore loss of its ability to function in translation, PCBP2 can mediate the switch from viral translation to RNA replication.

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Multimerization of poly(rC) binding protein 2 is required for translation initiation mediated by a viral IRES

Cited by 47 publications

References 32 publications

Identification of PCBP2, a facilitator of IRES-mediated translation, as a novel constituent of stress granules and processing bodies

Identification of PCBP2, a facilitator of IRES-mediated translation, as a novel constituent of stress granules and processing bodies

Evidence of reciprocal tertiary interactions between conserved motifs involved in organizing RNA structure essential for internal initiation of translation

Cellular Protein Modification by Poliovirus: the Two Faces of Poly(rC)-Binding Protein

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