Evidence that poly(A) binding protein C1 binds nuclear pre-mRNA poly(A) tails

Department of Biochemistry and Biophysics , University of Rochester, Rochester, New York, United States
Molecular and Cellular Biology (Impact Factor: 4.78). 05/2006; 26(8):3085-97. DOI: 10.1128/MCB.26.8.3085-3097.2006
Source: PubMed


In mammalian cells, poly(A) binding protein C1 (PABP C1) has well-known roles in mRNA translation and decay in the cytoplasm. However, PABPC1 also shuttles in and out of the nucleus, and its nuclear function is unknown. Here, we show that PABPC1, like the major nuclear poly(A) binding protein PABPN1, associates with nuclear pre-mRNAs that are polyadenylated and intron containing. PABPC1 does not bind nonpolyadenylated histone mRNA, indicating that the interaction of PABPC1 with pre-mRNA requires a poly(A) tail. Consistent with this conclusion, UV cross-linking results obtained using intact cells reveal that PABPC1 binds directly to pre-mRNA poly(A) tails in vivo. We also show that PABPC1 immunopurifies with poly(A) polymerase, suggesting that PABPC1 is acquired by polyadenylated transcripts during poly(A) tail synthesis. Our findings demonstrate that PABPC1 associates with polyadenylated transcripts earlier in mammalian mRNA biogenesis than previously thought and offer insights into the mechanism by which PABPC1 is recruited to newly synthesized poly(A). Our results are discussed in the context of pre-mRNA processing and stability and mRNA trafficking and the pioneer round of translation.

Download full-text


Available from: Fabrice Lejeune, Sep 06, 2015
6 Reads
  • Source
    • "PAP slowly adds A residues to the new 3′ end until a length sufficient for poly(A)-binding protein nuclear 1 (PABPN1) association is created. PABPN1 association with short poly(A) tails stimulates the rate of polyadenylation by PAP (Kuhn et al., 2009), culminating in a poly(A) tail of roughly 200–250 residues to which both PABPN1 and its largely cytoplasmic counterpart PABPC1 are bound in the nucleus (Hosoda et al., 2006). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mammalian-cell messenger RNAs (mRNAs) are generated in the nucleus from precursor RNAs (pre-mRNAs, which often contain one or more introns) that are complexed with an array of incompletely inventoried proteins. During their biogenesis, pre-mRNAs and their derivative mRNAs are subject to extensive cis-modifications. These modifications promote the binding of distinct polypeptides that mediate a diverse array of functions needed for mRNA metabolism, including nuclear export, inspection by the nonsense-mediated mRNA decay (NMD) quality-control machinery, and synthesis of the encoded protein product. Ribonucleoprotein complex (RNP) remodeling through the loss and gain of protein constituents before and after pre-mRNA splicing, during mRNA export, and within the cytoplasm facilitates NMD, ensuring integrity of the transcriptome. Here we review the mRNP rearrangements that culminate in detection and elimination of faulty transcripts by mammalian-cell NMD.
    Moleculer Cells 01/2014; 37(1):1-8. DOI:10.14348/molcells.2014.2193 · 2.09 Impact Factor
  • Source
    • "Although several mitochondrial proteins have been shown previously to bind RNA and poly(A) sequences in mitochondria (43–45), none binds poly(A) tails preferentially. Of the four human PABPC genes, three (PABPC1, PABPC3 and PABPC4) are established polyA-binding proteins (46–48); however, none has a predicted N-terminal MTS based on the annotated first AUG codon, or downstream AUG codon, and only PABPC5 predicts a dATI-dependent mitochondrial isoform. Several lines of evidence provide support for the proposed dATI-mediated mitochondrial isoform of PABPC5. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Alternative translation initiation (ATI) is a mechanism of producing multiple proteins from a single transcript, which in some cases regulates trafficking of proteins to different cellular compartments, including mitochondria. Application of a genome-wide computational screen predicts a cryptic mitochondrial targeting signal for 126 proteins in mouse and man that is revealed when an AUG codon located downstream from the canonical initiator methionine codon is used as a translation start site, which we term downstream ATI (dATI). Experimental evidence in support of dATI is provided by immunoblotting of endogenous truncated proteins enriched in mitochondrial cell fractions or of co-localization with mitochondria using immunocytochemistry. More detailed cellular localization studies establish mitochondrial targeting of a member of the cytosolic poly(A) binding protein family, PABPC5, and of the RNA/DNA helicase PIF1α. The mitochondrial isoform of PABPC5 co-immunoprecipitates with the mitochondrial poly(A) polymerase, and is markedly reduced in abundance when mitochondrial DNA and RNA are depleted, suggesting it plays a role in RNA metabolism in the organelle. Like PABPC5 and PIF1α, most of the candidates identified by the screen are not currently annotated as mitochondrial proteins, and so dATI expands the human mitochondrial proteome.
    Nucleic Acids Research 12/2012; 41(4). DOI:10.1093/nar/gks1347 · 9.11 Impact Factor
  • Source
    • "PABPC1 is a cytoplasmic protein involved in mRNA translation initiation and stability 49-52. When shuttled to the nucleus, PABPC1 engages in nuclear RNA biogenesis 50,53,54. In this study, we found that PABPC1 also binds to the 9-nt core of PAN MRE-II. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Kaposi sarcoma-associated herpesvirus (KSHV) ORF57, also known as Mta (mRNA transcript accumulation), enhances viral intron-less transcript accumulation and promotes splicing of intron-containing viral RNA transcripts. In this study, we identified KSHV PAN, a long non-coding polyadenylated nuclear RNA as a main target of ORF57 by a genome-wide CLIP (cross-linking and immunoprecipitation) approach. KSHV genome lacking ORF57 expresses only a minimal amount of PAN. In cotransfection experiments, ORF57 alone increased PAN expression by 20-30-fold when compared to vector control. This accumulation function of ORF57 was dependent on a structured RNA element in the 5' PAN, named MRE (Mta responsive element), but not much so on an ENE (expression and nuclear retention element) in the 3' PAN previously reported by other studies. We showed that the major function of the 5' PAN MRE is increasing the RNA half-life of PAN in the presence of ORF57. Further mutational analyses revealed a core motif consisting of 9 nucleotides in the MRE-II , which is responsible for ORF57 interaction and function. The 9-nt core in the MRE-II also binds cellular PABPC1, but not the E1B-AP5 which binds another region of the MRE-II. In addition, we found that PAN RNA is partially exportable in the presence of ORF57. Together, our data provide compelling evidence as to how ORF57 functions to accumulate a non-coding viral RNA in the course of virus lytic infection.
    International journal of biological sciences 10/2011; 7(8):1145-60. · 4.51 Impact Factor
Show more