Drosophila Trf4-1 involvement in mRNA and primary miRNA transcription
ABSTRACT: Drosophila Trf4-1 is a polyadenylation polymerase or terminal nucleotidyl transferase (PAP/TENT) that has been reported to add poly(A) tails to snRNAs in nucleus or mRNAs in cytoplasm. Here, we found that the loss of Trf4-1 resulted in the reduction of mRNAs and primary miRNAs (pri-miRNAs) in both Drosophila S2 cells and adult flies. Interestingly, the role of Trf4-1 in transcription is independent of its PAP/TENT activity. Moreover, using the chromatin immunoprecipitation assay, we uncovered that the loss of Trf4-1 led to abnormal RNA polymerase II accumulation and reduced H3K4me3 binding in promoter regions. Overall design: Small RNA profile of RNAi based knock-down cells
Project description:The large subunit of Saccharomyces cerevisiae DNA polymerase epsilon, Pol2, comprises two essential functions. The N terminus has essential DNA polymerase activity. The C terminus is also essential, but its function is unknown. We report here that the C-terminal domain of Pol2 interacts with polymerase sigma (Pol sigma), a recently identified, essential nuclear nucleotidyl transferase encoded by two redundant genes, TRF4 and TRF5. This interaction is functional, since Pol sigma stimulates the polymerase activity of the Pol epsilon holoenzyme significantly. Since Trf4 is required for sister chromatid cohesion as well as for completion of S phase and repair, the interaction suggested that Pol epsilon, like Pol sigma, might form a link between the replication apparatus and sister chromatid cohesion and/or repair machinery. We present evidence that pol2 mutants are defective in sister chromatid cohesion. In addition, Pol2 interacts with SMC1, a subunit of the cohesin complex, and with ECO1/CTF7, required for establishing sister chromatid cohesion; and pol2 mutations act synergistically with smc1 and scc1. We also show that trf5 Delta mutants, like trf4 Delta mutants, are defective in DNA repair and sister chromatid cohesion.
Project description:The Caenorhabditis elegans germ-line development defective (GLD)-2-GLD-3 complex up-regulates the expression of genes required for meiotic progression. GLD-2-GLD-3 acts by extending the short poly(A) tail of germ-line-specific mRNAs, switching them from a dormant state into a translationally active state. GLD-2 is a cytoplasmic noncanonical poly(A) polymerase that lacks the RNA-binding domain typical of the canonical nuclear poly(A)-polymerase Pap1. The activity of C. elegans GLD-2 in vivo and in vitro depends on its association with the multi-K homology (KH) domain-containing protein, GLD-3, a homolog of Bicaudal-C. We have identified a minimal polyadenylation complex that includes the conserved nucleotidyl-transferase core of GLD-2 and the N-terminal domain of GLD-3, and determined its structure at 2.3-Å resolution. The structure shows that the N-terminal domain of GLD-3 does not fold into the predicted KH domain but wraps around the catalytic domain of GLD-2. The picture that emerges from the structural and biochemical data are that GLD-3 activates GLD-2 both indirectly by stabilizing the enzyme and directly by contributing positively charged residues near the RNA-binding cleft. The RNA-binding cleft of GLD-2 has distinct structural features compared with the poly(A)-polymerases Pap1 and Trf4. Consistently, GLD-2 has distinct biochemical properties: It displays unusual specificity in vitro for single-stranded RNAs with at least one adenosine at the 3' end. GLD-2 thus appears to have evolved specialized nucleotidyl-transferase properties that match the 3' end features of dormant cytoplasmic mRNAs.
Project description:Poly(A) polymerase (PAP) has a role in two processes, polyadenylation of mRNA precursors in the nucleus and translational control of certain mRNAs by cytoplasmic elongation of their poly(A) tails, particularly during early development. It was found recently that at least three different PAP genes exist in mammals, encoding several PAP isoforms. The in vivo specificity of function of each PAP isoform currently is unknown. Here, we analyse PAP function in Drosophila: We show that a single PAP isoform exists in Drosophila that is encoded by the hiiragi gene. This single Drosophila PAP is active in specific polyadenylation in vitro and is involved in both nuclear and cytoplasmic polyadenylation in vivo. Therefore, the same PAP can be responsible for both processes. In addition, in vivo overexpression of PAP does not affect poly(A) tail length during nuclear polyadenylation, but leads to a dramatic elongation of poly(A) tails and a loss of specificity during cytoplasmic polyadenylation, resulting in embryonic lethality. This demonstrates that regulation of the PAP level is essential for controlled cytoplasmic polyadenylation and early development.
Project description:A key question in nuclear RNA surveillance is how target RNAs are recognized. To address this, we identified in vivo binding sites for nuclear RNA surveillance factors, Nrd1, Nab3 and the Trf4/5–Air1/2–Mtr4 polyadenylation (TRAMP) complex poly(A) polymerase Trf4, by UV crosslinking. Hit clusters were reproducibly found over known binding sites on small nucleolar RNAs (snoRNAs), pre-mRNAs and cryptic, unstable non-protein-coding RNAs (ncRNAs) ('CUTs'), along with ~642 predicted long anti-sense ncRNAs (asRNAs), ~178 intergenic ncRNAs and, surprisingly, ~1384 mRNAs. Five putative asRNAs tested were confirmed to exist and were stabilized by loss of Nrd1, Nab3 or Trf4. Mapping of micro-deletions and substitutions allowed clear definition of preferred, in vivo Nab3 and Nrd1 binding sites. Nrd1 and Nab3 were believed to be Pol II specific but, unexpectedly, bound many oligoadenylated Pol III transcripts, predominately pre-tRNAs. Depletion of Nrd1 or Nab3 stabilized tested Pol III transcripts and their oligoadenylation was dependent on Nrd1–Nab3 and TRAMP. Surveillance targets were enriched for non-encoded A-rich tails. These were generally very short (1–5 nt), potentially explaining why adenylation destabilizes these RNAs while stabilizing mRNAs with long poly(A) tails.
Project description:During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity is apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association shows co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduces Mtr4 recruitment and increases RNA abundance for mRNAs specifically showing high Trf5 binding.
Project description:The S-M checkpoint delays mitosis until DNA replication is complete; cells defective in this checkpoint lose viability when DNA replication is inhibited. This inviability can be suppressed in fission yeast by overexpression of Cid1 or the related protein Cid13. Fission yeast contain six cid1/cid13-like genes, whereas budding yeast has just two, TRF4 and TRF5. Trf4 and Trf5 were recently reported to comprise an essential DNA polymerase activity required for the establishment of sister chromatid cohesion. In contrast, we find that Cid1 is not a DNA polymerase but instead uses RNA substrates and has poly(A) polymerase activity. Unlike the previously characterized yeast poly(A) polymerase, which is a nuclear enzyme, Cid1 and Cid13 are constitutively cytoplasmic. Cid1 has a degree of substrate specificity in vitro, consistent with the notion that it targets a subset of cytoplasmic mRNAs for polyadenylation in vivo, hence increasing their stability and/or efficiency of translation. Preferred Cid1 targets presumably include mRNAs encoding components of the S-M checkpoint, whereas Cid13 targets are likely to be involved in dNTP metabolism. Cytoplasmic polyadenylation is known to be an important regulatory mechanism during early development in animals. Our findings in yeast suggest that this level of gene regulation is of more general significance in eukaryotic cells.
Project description:Tumor protein D52 (TPD52) is an oncogene amplified and overexpressed in various cancers. Tumor-suppressive microRNA-449a and microRNA-34a (miR-449a/34a) were recently reported to inhibit breast cancer cell migration and invasion via targeting TPD52. However, the upstream events are not clearly defined. Star-PAP is a non-canonical poly (A) polymerase which could regulate the expression of many miRNAs and mRNAs, but its biological functions are not well elucidated. The present study aimed to explore the regulative roles of Star-PAP in miR-449a/34a and TPD52 expression in breast cancer. We observed a negative correlation between the expression of TPD52 and Star-PAP in breast cancer. Overexpression of Star-PAP inhibited TPD52 expression, while endogenous Star-PAP knockdown led to increased TPD52. Furthermore, RNA immunoprecipitation assay suggested that Star-PAP could not bind to TPD52, independent of the 3'-end processing. RNA pull-down assay showed that Star-PAP could bind to 3'region of miR-449a. In line with these results, blunted cell proliferation or cell apoptosis caused by Star-PAP was rescued by overexpression of TPD52 or downregulation of miR-449a/34a. Our findings identified that Star-PAP regulates TPD52 by modulating miR-449a/34a, which may be an important molecular mechanism underlying the tumorigenesis of breast cancer and provide a rational therapeutic target for breast cancer treatment.
Project description:Base pairing between the 5' end of U7 small nuclear RNA (snRNA) and the histone downstream element (HDE) in replication-dependent histone pre-mRNAs is the key event in 3'-end processing that leads to generation of mature histone mRNAs. We have cloned the Drosophila U7 snRNA and demonstrated that it is required for histone pre-mRNA 3'-end processing in a Drosophila nuclear extract. The 71-nt Drosophila U7 snRNA is encoded by a single gene that is embedded in the direct orientation in an intron of the Eip63E gene. The U7 snRNA gene contains conserved promoter elements typical of other Drosophila snRNA genes, and the coding sequence is followed by a 3' box indicating that the Drosophila U7 snRNA gene is an independent transcription unit. Drosophila U7 snRNA contains a trimethyl-guanosine cap at the 5' end and a putative Sm-binding site similar to the unique Sm-binding site found in other U7 snRNAs. Drosophila U7 snRNA is approximately 10 nt longer than mammalian U7 snRNAs because of an extended 5' sequence and has only a limited potential to form a stem-loop structure near the 3' end. The extended 5' end of Drosophila U7 snRNA can base pair with the HDE in all five Drosophila histone pre-mRNAs. Blocking the 5' end of the U7 snRNA with a complementary oligonucleotide specifically blocks processing of a Drosophila histone pre-mRNA. Changes in the HDE that abolish or decrease processing efficiency result in a reduced ability to recruit U7 snRNA to the pre-mRNA.
Project description:Nuclear poly(A) polymerase (PAP) polyadenylates nascent mRNAs, promoting their nuclear export, stability, and translation, while the related cytoplasmic polymerase GLD-2 activates translation of deadenylated mRNAs. Here we characterize the biochemical activity of fission yeast Schizosaccharomyces pombe Cid1, a putative cytoplasmic PAP implicated in cell cycle checkpoint controls. Surprisingly, Cid1 has robust poly(U) polymerase activity in vitro, especially when isolated in native multiprotein complexes. Furthermore, we found that upon S-phase arrest, the 3' ends of actin mRNAs were posttranscriptionally uridylated in a Cid1-dependent manner. Finally, Hs2 (ZCCHC6), a human ortholog of Cid1, shows similar activity. These data suggest that uridylation of mRNA forms the basis of an evolutionarily conserved mechanism of gene regulation.
Project description:Post-transcriptional 3' end addition of nucleotides is important in a variety of RNA decay pathways. We have examined the 3' end addition of nucleotides during the decay of the Hsp70 mRNA and a corresponding reporter RNA in Drosophila S2 cells by conventional sequencing of cDNAs obtained after mRNA circularization and by deep sequencing of dedicated libraries enriched for 3' decay intermediates along the length of the mRNA. Approximately 5%-10% of 3' decay intermediates carried nonencoded oligo(A) tails with a mean length of 2-3 nucleotides. RNAi experiments showed that the oligoadenylated RNA fragments were intermediates of exosomal decay and the noncanonical poly(A) polymerase Trf4-1 was mainly responsible for A addition. A hot spot of A addition corresponded to an intermediate of 3' decay that accumulated upon inhibition of decapping, and knockdown of Trf4-1 increased the abundance of this intermediate, suggesting that oligoadenylation facilitates 3' decay. Oligoadenylated 3' decay intermediates were found in the cytoplasmic fraction in association with ribosomes, and fluorescence microscopy revealed a cytoplasmic localization of Trf4-1. Thus, oligoadenylation enhances exosomal mRNA degradation in the cytoplasm.