Naoyuki Kataoka

Functional Association of the Microprocessor Complex with the Spliceosome

ABSTRACT The majority of human microRNAs (miRNAs) are located in the introns of other genes (A. Rodriguez, S. Griffiths-Jones, J. L. Ashurst, and A. Bradley, Genome Res. 14:1902-1910, 2004). Based on the discovery that artificial insertion of pre-miRNAs in introns did not hamper mRNA production and that the miRNA-harboring introns were spliced more slowly than the adjacent introns, a model was previously proposed in which Drosha crops the pre-miRNA and the two cropped fragments from the pre-mRNA are subsequently trans spliced (Y. K. Kim and V. N. Kim, EMBO J. 26:775-783, 2007). However, the molecular basis for this model was not elucidated. To analyze the molecular mechanism of intronic miRNA processing, we developed an in vitro system in which both pre-miRNA processing and mRNA splicing are detected simultaneously. Our analysis using this system showed that pre-miRNA cropping from the pre-mRNA could occur kinetically faster than splicing. Glycerol gradient sedimentation experiments revealed that part of the pre-miRNA was cofractionated with the spliceosome. Furthermore, coimmunoprecipitation experiments with an anti-Drosha antibody demonstrated that Drosha was associated not only with the cropping products but also with a Y-shaped branch intron and a Y-shaped splicing intermediate. These results provide a molecular basis for the postulated existence of a pathway in which the Microprocessor complex becomes associated with the spliceosome, pre-miRNA cropping occurs prior to splicing, and trans splicing takes place between the cropped products.

Chemical treatment enhances skipping of a mutated exon in the dystrophin gene

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by a loss of the dystrophin protein. Control of dystrophin mRNA splicing to convert severe DMD to a milder phenotype is attracting much attention. Here we report a dystrophinopathy patient who has a point mutation in exon 31 of the dystrophin gene. Although the mutation generates a stop codon, a small amount of internally deleted, but functional, dystrophin protein is produced in the patient cells. An analysis of the mRNA reveals that the mutation promotes exon skipping and restores the open reading frame of dystrophin. Presumably, the mutation disrupts an exonic splicing enhancer and creates an exonic splicing silencer. Therefore, we searched for small chemicals that enhance exon skipping, and found that TG003 promotes the skipping of exon 31 in the endogenous dystrophin gene in a dose-dependent manner and increases the production of the dystrophin protein in the patients cells.

Stress-responsive maturation of Clk1/4 pre-mRNAs promotes phosphorylation of SR splicing factor.

A nuclear pool of partially spliced Clk1/4 pre-mRNAs matures in response to stress and induces SR protein phosphorylation and activation.

Rectifier of aberrant mRNA splicing recovers tRNA modification in familial dysautonomia

Significance Familial dysautonomia (FD) is caused by missplicing of the IκB kinase complex-associated protein (IKAP) gene, which results in the skipping of exon 20, especially in neurons. FD would be treatable if exon 20 inclusion were increased correctly to reestablish correct splicing. Here, we have established a dual-color splicing reporter that recapitulates FD-type splicing. By using this reporter, we have identified a small chemical compound, named rectifier of aberrant splicing (RECTAS), that rectifies the aberrant splicing of FD. RECTAS promotes both exon 20 inclusion and the product IKAP expression in cells of patients with FD. Furthermore, we have demonstrated that modification levels of wobble uridine residues of several tRNAs are reduced in FD cells and that RECTAS can recover not only tRNA modifications but also cell viability of FD cells. Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by missplicing of exon 20, resulting from an intronic mutation in the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene encoding IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1). A newly established splicing reporter assay allowed us to visualize pathogenic splicing in cells and to screen small chemicals for the ability to correct the aberrant splicing of IKBKAP. Using this splicing reporter, we screened our chemical libraries and identified a compound, rectifier of aberrant splicing (RECTAS), that rectifies the aberrant IKBKAP splicing in cells from patients with FD. Here, we found that the levels of modified uridine at the wobble position in cytoplasmic tRNAs are reduced in cells from patients with FD and that treatment with RECTAS increases the expression of IKAP and recovers the tRNA modifications. These findings suggest that the missplicing of IKBKAP results in reduced tRNA modifications in patients with FD and that RECTAS is a promising therapeutic drug candidate for FD.

Herpesvirus protein ICP27 switches PML isoform by altering mRNA splicing

Viruses use alternative splicing to produce a broad series of proteins from small genomes by utilizing the cellular splicing machinery. Since viruses use cellular RNA binding proteins for viral RNA processing, it is presumable that the splicing of cellular pre-mRNAs is affected by viral infection. Here, we showed that herpes simplex virus type 2 (HSV-2) modifies the expression of promyelocytic leukemia (PML) isoforms by altering pre-mRNA splicing. Using a newly developed virus-sensitive splicing reporter, we identified the viral protein ICP27 as an alternative splicing regulator of PML isoforms. ICP27 was found to bind preferentially to PML pre-mRNA and directly inhibit the removal of PML intron 7a in vitro. Moreover, we demonstrated that ICP27 functions as a splicing silencer at the 3′ splice site of the PML intron 7a. The switching of PML isoform from PML-II to PML-V as induced by ICP27 affected HSV-2 replication, suggesting that the viral protein modulates the splicing code of cellular pre-mRNA(s) governing virus propagation.

Isolation and characterization of post-splicing lariat–intron complexes

Pre-mRNA splicing occurs in a large complex spliceosome. The steps of both spliceosome assembly and splicing reaction have been extensively analyzed, and many of the factors involved have been identified. However, the post-splicing intron turnover process, especially in vertebrates, remains to be examined. In this paper, we developed a two-tag affinity purification method for purifying lariat intron RNA–protein complexes obtained from an in vitro splicing reaction. Glycerol gradient sedimentation analyses revealed that there are at least two forms of post-splicing intron complexes, which we named the ‘Intron Large (IL)’ and the ‘Intron Small (IS)’ complexes. The IL complex contains U2, U5 and U6 snRNAs and other protein splicing factors, whereas the IS complex contains no such U snRNAs or proteins. We also showed that TFIP11, a human homolog of yeast Ntr1, is present in the IL complex and the TFIP11 mutant protein, which lacks the interaction domain with hPrp43 protein, caused accumulation of the IL complex and reduction of IS complex formation in vitro. Taken together, our results strongly suggest that TFIP11 in cooperation with hPrp43 mediates the transition from the IL complex to the IS complex, leading to efficient debranching and turnover of excised introns.

Combination of Clk family kinase and SRp75 modulates alternative splicing of Adenovirus E1A

SR proteins are non‐snRNP splicing factors harbouring a domain rich in Arg‐Ser repeats, which are extensively phosphorylated by several kinases. We performed a comparative study of different SR kinases, including SRPK, Clk, PRP4 and DYRK, and found that only Clks efficiently altered 5′ splice site selection of Adenovirus E1A. The phosphorylation state of SR proteins was examined using a phospho‐SR specific antibody mAb1H4 and a 75 kDa protein was most evidently hyperphosphorylated by Clks. Administration of TG003, a specific inhibitor for the Clk family members, specifically and rapidly induced dephosphorylation of 75 kDa SR protein. Imaging with mRFP‐SRp75 in living cells revealed that its nuclear distribution was rapidly altered upon inhibition of the Clk activity by TG003. Co‐transfection experiments demonstrated that HA‐tagged SRp75 was hyperphosphorylated by Clk family members, but not by other SR kinases. These results indicate that Clks specifically hyperphosphorylate SRp75. Furthermore, SRp75 over‐expression promoted the selection of 12S 5′ splice site in E1A pre‐mRNA, which is stimulated by co‐expression of Clks. These results suggest that the specific combination of SR protein and SR kinase plays a distinct role in alternative splicing through dynamic balance of phosphorylation.

Ce-Y14 and MAG-1, components of the exon-exon junction complex, are required for embryogenesis and germline sexual switching in Caenorhabditis elegans.

Y14 is a component of the splicing-dependent exon-exon junction complex (EJC) and is involved in the mRNA quality control system called nonsense-mediated mRNA decay. It has recently been shown that together with another EJC component, Mago, the Drosophila homologue DmY14/Tsunagi is required for proper localization of oskar mRNA during oogenesis, a process critical for posterior formation in Drosophila development. Here we show that the nematode Caenorhabditis elegans Ce-Y14 and MAG-1 (Mago homologue) are required for late embryogenesis and proper germline sexual differentiation. Like in other organisms, Ce-Y14 preferentially binds to spliced mRNA and specifically interacts with MAG-1. Consistent with the evolutionarily conserved interaction between Y14 and Mago homologues, suppression of Ce-Y14 by RNAi resulted in the same phenotypes as those caused by RNAi of mag-1 lethality during late embryogenesis and masculinization of the adult hermaphrodite germline. Our results demonstrate that the evolutionarily conserved interaction between two EJC components, Ce-Y14 and MAG-1, has critical developmental roles in C. elegans.

SR proteins preferentially associate with mRNAs in the nucleus and facilitate their export to the cytoplasm

Different classes of RNA are exported to the cytoplasm by distinct mechanisms. Each class of RNA forms distinct complexes with nuclear proteins prior to its export to the cytoplasm. In our attempt to obtain comprehensive information of protein factors that specifically associate with mRNAs in the nucleus, we performed in vivo UV‐crosslinking analysis after microinjection of various RNAs into Xenopus oocyte nucleus. We found a group of proteins preferentially crosslinked to mRNAs. Immunoprecipitation experiments revealed that some of the crosslinked signals corresponded to SR (serine/arginine‐rich) proteins, a family of essential RNA‐binding proteins involved in pre‐mRNA splicing. It was previously suggested that some members of SR protein family are involved in export of a specific intronless mRNA, histone H2A mRNA and some spliced mRNAs. However, it is still to be clarified if SR proteins are involved in export of general mRNAs, especially general intronless mRNAs that do not contain specific RNA export elements. When we microinjected an antibody against SR proteins into the nucleus, export of mRNAs was severely inhibited, regardless of whether the mRNAs were produced via pre‐mRNA splicing or not, whereas export of other RNAs was not affected. These results unequivocally showed that SR proteins are involved in export of both general intronless and spliced mRNAs.

Preparation of Efficient Splicing Extracts From Whole Cells, Nuclei, and Cytoplasmic Fractions

Pre-mRNA (messenger RNA) splicing is an essential step for gene expression in higher eukaryotes. Splicing reactions have been well studied in vitro using extracts prepared from cultured cells. We describe protocols for the preparation of splicing-competent extracts from whole cells, nuclei, and cytoplasmic fractions. The nuclear and whole-cell extracts are fully active in splicing, while S100 extracts are able to support splicing only when SR (Serine/Arginine-rich) proteins are supplied. The simple method described here to prepare splicing active extracts from whole cells is particularly useful in studying pre-mRNA splicing in many different cell types.