Venkataraman, K., Brown, K.M. & Gilmartin, G.M. Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition. Genes Dev. 19, 1315-1327

Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, 05405, USA.
Genes & Development (Impact Factor: 10.8). 07/2005; 19(11):1315-27. DOI: 10.1101/gad.1298605
Source: PubMed


At least half of all human pre-mRNAs are subject to alternative 3' processing that may modulate both the coding capacity of the message and the array of post-transcriptional regulatory elements embedded within the 3' UTR. Vertebrate poly(A) site selection appears to rely primarily on the binding of CPSF to an A(A/U)UAAA hexamer upstream of the cleavage site and CstF to a downstream GU-rich element. At least one-quarter of all human poly(A) sites, however, lack the A(A/U)UAAA motif. We report that sequence-specific RNA binding of the human 3' processing factor CFI(m) can function as a primary determinant of poly(A) site recognition in the absence of the A(A/U)UAAA motif. CFI(m) is sufficient to direct sequence-specific, A(A/U)UAAA-independent poly(A) addition in vitro through the recruitment of the CPSF subunit hFip1 and poly(A) polymerase to the RNA substrate. ChIP analysis indicates that CFI(m) is recruited to the transcription unit, along with CPSF and CstF, during the initial stages of transcription, supporting a direct role for CFI(m) in poly(A) site recognition. The recognition of three distinct sequence elements by CFI(m), CPSF, and CstF suggests that vertebrate poly(A) site definition is mechanistically more similar to that of yeast and plants than anticipated.

Full-text preview

Available from:
  • Source
    • "In the first deletion construct (pRRpCeGFPWPRE25_1293), the complete promoter/enhancer region, including the TATA box, was removed, leaving only the core elements of pre-mRNA processing signals (cleavage and polyadenylation specificity factor [CPSF] and cleavage stimulating factor [CstF] recognition motifs). The second SIN vector (pRRpCeGFPWPRE25_1218) contained, in addition to the CPSF and the CstF recognition motifs, additional cis-acting elements including the U-rich region and CF Im recognition motif, involved in the regulation of mRNA polyadenylation [25,26]. When the SIN vector with the longer deletion, pRRpCeGFPWPRE25_1293, was used for transduction, the titers determined were approximately 20% of those obtained with the parental vector carrying an intact 3′LTR. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mouse mammary tumor virus (MMTV) is a complex, milk-born betaretrovirus, which preferentially infects dendritic cells (DC) in the gastrointestinal tract and then spreads to T and B lymphocytes and finally to the mammary gland. It is not clear how the prototypic betaretrovirus infects mucosal DCs and naive lymphocytes as these cells are considered to be non-proliferative. Studies of MMTV biology have been hampered by the difficulty of obtaining sufficient virus/vector titers after transfection of a molecular clone in cultured cells. To surmount this barrier we developed a novel MMTV-based vector system with a split genome design containing potent posttranscriptional regulatory functions. Using this system, vector particles were produced to markedly greater titers (>1000-fold) than those obtained previously. The titers (>106 transduction units /ml) were comparable to those achieved with lentiviral or gammaretroviral vectors. Importantly, the vector transduced the enhanced green fluorescence protein gene into the chromosomes of non-dividing cells, such as cells arrested at the G2/M phase of the cell cycle and unstimulated hematopoietic progenitor cells, at an efficiency similar to that obtained with the HIV-1-based vector. In contrast to HIV-1, MMTV transductions were not affected by knocking down the expression of a factor involved in nuclear import of the HIV-1 pre-integration complexes, TNPO3. In contrast to HIV-1, the MMTV-based vector did not preferentially integrate in transcription units. Additionally, no preference for integration near transcription start sites, the regions preferentially targeted by gammaretroviral vectors, was observed. The vector derived from MMTV exhibits a random integration pattern. Overall, the betaretroviral vector system should facilitate molecular virology studies of the prototypic betaretrovirus as well as studies attempting to elucidate fundamental cellular processes such as nuclear import pathways. Random integration in cycling and non-cycling cells may be applicable in unbiased gene delivery.
    Retrovirology 04/2014; 11(1):34. DOI:10.1186/1742-4690-11-34 · 4.19 Impact Factor
  • Source
    • "The 160 kDa subunit of the cleavage and polyadenylation specificity factor (CPSF) recognizes the PAS [17], while the N-terminal RNA recognition motif (RRM) from the 64 kDa subunit of the cleavage stimulatory factor (CstF) binds the DSE [18]. Cleavage factor I (CFIm) interacts with the UGUAN (N=A>U≥C/G) motif [19,20], which promotes the recruitment of CFIIm, poly(A) polymerase (PAP), poly(A) binding protein (PABP), transcriptional coactivator PC4, symplekin and carboxyterminal domain of the RNA polymerase II largest subunit (RNA pol II CTD) [21]. CPSF-73 is thought to perform pre-mRNA cleavage [22], before PAP catalyzes the poly(A) tail synthesis [21]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In eukaryotes, polyadenylation of pre-mRNA 3´ end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25) from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X) domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25) was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A) polymerase (EhPAP) that is responsible for the synthesis of the poly(A) tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A) polymerase, another member of the pre-mRNA 3´ end processing machinery in this protozoan parasite.
    PLoS ONE 08/2013; 8(6):e67977. DOI:10.1371/journal.pone.0067977 · 3.23 Impact Factor
  • Source
    • "Similar to other pre-mRNA cleavage and polyadenylation factors, CFIm is also co-transcriptionally recruited to genes (15). To determine whether Thoc5 regulates the efficiency of CFIm68 recruitment to genes, we performed a genome-wide ChIP-Seq analysis in which HeLa cells treated with siThoc5 or control siDsRed were subjected to ChIP using anti-CFIm68 antibodies. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The transcription-export complex (TREX) couples mRNA transcription, processing and nuclear export. We found that CFIm68, a large subunit of a heterotetrameric protein complex mammalian cleavage factor I (CFIm), which is implicated in alternative polyadenylation site choice, co-purified with Thoc5, a component of human TREX. Immunoprecipitation using antibodies against different components of TREX indicated that most likely both complexes interact via an interaction between Thoc5 and CFIm68. Microarray analysis using human HeLa cells revealed that a subset of genes was differentially expressed on Thoc5 knockdown. Notably, the depletion of Thoc5 selectively attenuated the expression of mRNAs polyadenylated at distal, but not proximal, polyadenylation sites, which phenocopied the depletion of CFIm68. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) indicated that CFIm68 preferentially associated with the 5′ regions of genes; strikingly, the 5′ peak of CFIm68 was significantly and globally reduced on Thoc5 knockdown. We suggest a model in which human Thoc5 controls polyadenylation site choice through the co-transcriptional loading of CFIm68 onto target genes.
    Nucleic Acids Research 05/2013; 41(14). DOI:10.1093/nar/gkt414 · 9.11 Impact Factor
Show more