EnABLing microprocessor for apoptosis.

The Microprocessor complex consisting of DROSHA (a type III ribonuclease) and DGCR8 (DiGeorge syndrome critical region gene 8-encoded RNA binding protein) recognizes and cleaves the precursor microRNA hairpin (pre-miRNA) from the primary microRNA transcript (pri-miRNA). The Abelson tyrosine kinase 1 (ABL) phosphorylates DGCR8 to stimulate the cleavage of a subset of pro-apoptotic pri-miRNAs, thus expanding the nuclear functions of ABL to include regulation of RNA processing.

RBM4 promotes neuronal differentiation and neurite outgrowth by modulating Numb isoform expression.

RBM4 participates in cell differentiation by regulating tissue-specific alternative pre-mRNA splicing. RBM4 also has been implicated in neurogenesis in the mouse embryonic brain. Using mouse embryonal carcinoma P19 cells as a neural differentiation model, we observed a temporal correlation between RBM4 expression and a change in splicing isoforms of Numb, a cell-fate determination gene. Knockdown of RBM4 affected the inclusion/exclusion of exons 3 and 9 of Numb in P19 cells. RBM4-deficient embryonic mouse brain also exhibited aberrant splicing of Numb pre-mRNA. Using a splicing reporter minigene assay, we demonstrated that RBM4 promoted exon 3 inclusion and exon 9 exclusion. Moreover, we found that RBM4 depletion reduced the expression of the proneural gene Mash1, and such reduction was reversed by an RBM4-induced Numb isoform containing exon 3 but lacking exon 9. Accordingly, induction of ectopic RBM4 expression in neuronal progenitor cells increased Mash1 expression and promoted cell differentiation. Finally, we found that RBM4 was also essential for neurite outgrowth from cortical neurons in vitro. Neurite outgrowth defects of RBM4-depleted neurons were rescued by RBM4-induced exon 9-lacking Numb isoforms. Therefore our findings indicate that RBM4 modulates exon selection of Numb to generate isoforms that promote neuronal cell differentiation and neurite outgrowth.

Proteomics studies of the interactome of RNA polymerase II C-terminal repeated domain.

BACKGROUND: Eukaryotic RNA polymerase II contains a C-terminal repeated domain (CTD) consisting of 52 consensus heptad repeats of Y1S2P3T4S5P6S7 that mediate interactions with many cellular proteins to regulate transcription elongation, RNA processing and chromatin structure. A number of CTD-binding proteins have been identified and the crystal structures of several protein-CTD complexes have demonstrated considerable conformational flexibility of the heptad repeats in those interactions. Furthermore, phosphorylation of the CTD at tyrosine, serine and threonine residues can regulate the CTD-protein interactions. Although the interactions of CTD with specific proteins have been elucidated at the atomic level, the capacity and specificity of the CTD-interactome in mammalian cells is not yet determined. RESULTS: A proteomic study was conducted to examine the mammalian CTD-interactome. We utilized six synthetic peptides each consisting of four consensus CTD-repeats with different combinations of serine and tyrosine phosphorylation as affinity-probes to pull-down nuclear proteins from HeLa cells. The pull-down fractions were then analyzed by MUDPIT mass spectrometry, which identified 100 proteins with the majority from the phospho-CTD pull-downs. Proteins pulled-down by serine-phosphorylated CTD-peptides included those containing the previously defined CTD-interacting domain (CID). Using SILAC mass spectrometry, we showed that the in vivo interaction of RNA polymerase II with the mammalian CID-containing RPRD1B is disrupted by CID mutation. We also showed that the CID from four mammalian proteins interacted with pS2-phosphorylated but not pY1pS2-doubly phosphorylated CTD-peptides. However, we also found proteins that were preferentially pulled-down by pY1pS2- or pY1pS5-doubly phosphorylated CTD-peptides. We prepared an antibody against tyrosine phosphorylated CTD and showed that ionizing radiation (IR) induced a transient increase in CTD tyrosine phosphorylation by immunoblotting. Combining SILAC and IMAC purification of phospho-peptides, we found that IR regulated the phosphorylation at four CTD tyrosine sites in different ways. CONCLUSION: Upon phosphorylation, the 52 repeats of the CTD have the capacity to generate a large number of binding sites for cellular proteins. This study confirms previous findings that serine phosphorylation stimulates whereas tyrosine phosphorylation inhibits the protein-binding activity of the CTD. However, tyrosine phosphorylation of the CTD can also stimulate other CTD-protein interactions. The CTD-peptide affinity pull-down method described here can be adopted to survey the mammalian CTD-interactome in various cell types and under different biological conditions.

The kinase ABL phosphorylates the microprocessor subunit DGCR8 to stimulate primary microRNA processing in response to DNA damage.

The DNA damage response network stimulates microRNA (miRNA) biogenesis to coordinate repair, cell cycle checkpoints, and apoptosis. The multistep process of miRNA biogenesis involves the cleavage of primary miRNAs by the microprocessor complex composed of the ribonuclease Drosha and the RNA binding protein DGCR8. We found that the tyrosine kinase ABL phosphorylated DGCR8, a modification that was required for the induction of a subset of miRNAs after DNA damage. Focusing on the miR-34 family, ABL stimulated the production of miR-34c, but not miR-34a, through Drosha/DGCR8-dependent processing of primary miR-34c (pri-miR-34c). This miRNA-selective effect of ABL required the sequences flanking the precursor miR-34c (pre-miR-34c) stem-loop. In pri-miRNA processing, DGCR8 binds the pre-miR stem-loop and recruits Drosha to the miRNA. RNA cross-linking assays showed that DGCR8 and Drosha interacted with pri-miR-34c, but we found an inverse correlation between ABL-stimulated processing and DGCR8 association with pri-miR-34c. When coexpressed in HEK293T cells, ABL phosphorylated DGCR8 at Tyr(267). Ectopic expression of a Y267F-DGCR8 mutant reduced the recruitment of Drosha to pri-miR-34c and prevented ABL or Drosha from stimulating the processing of pri-miR-34c. In mice engineered to express a nuclear import-defective mutant of ABL, miR-34c, but not miR-34a, expression was reduced in the kidney, and apoptosis of the renal epithelial cells was impaired in response to cisplatin. These results reveal a new pathway in the DNA damage response wherein ABL-dependent tyrosine phosphorylation of DGCR8 stimulates the processing of selective primary miRNAs.