Hitoshi Suzuki

A vertebrate RNA-binding protein Fox-1 regulates tissue-specific splicing via the pentanucleotide GCAUG

Alternative splicing is one of the central mechanisms that regulate eukaryotic gene expression. Here we report a tissue‐specific RNA‐binding protein, Fox‐1, which regulates alternative splicing in vertebrates. Fox‐1 bound specifically to a pentanucleotide GCAUG in vitro. In zebrafish and mouse, fox‐1 is expressed in heart and skeletal muscles. As candidates for muscle‐specific targets of Fox‐1, we considered two genes, the human mitochondrial ATP synthase γ‐subunit gene (F1γ) and the rat α‐actinin gene, because their primary transcripts contain several copies of GCAUG. In transfection experiments, Fox‐1 induced muscle‐specific exon skipping of the F1γ gene via binding to GCAUG sequences upstream of the regulated exon. Fox‐1 also regulated mutually exclusive splicing of the α‐actinin gene, antagonizing the repressive effect of polypyrimidine tract‐binding protein (PTB). It has been reported that GCAUG is essential for the alternative splicing regulation of several genes including fibronectin. We found that Fox‐1 promoted inclusion of the fibronectin EIIIB exon. Thus, we conclude that Fox‐1 plays key roles in both positive and negative regulation of tissue‐specific splicing via GCAUG.

Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing

Besides linear RNAs, pre-mRNA splicing generates three forms of RNAs: lariat introns, Y-structure introns from trans-splicing, and circular exons through exon skipping. To study the persistence of excised introns in total cellular RNA, we used three Escherichia coli 3′ to 5′ exoribonucleases. Ribonuclease R (RNase R) thoroughly degrades the abundant linear RNAs and the Y-structure RNA, while preserving the loop portion of a lariat RNA. Ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase) also preserve the lariat loop, but are less efficient in degrading linear RNAs. RNase R digestion of the total RNA from human skeletal muscle generates an RNA pool consisting of lariat and circular RNAs. RT–PCR across the branch sites confirmed lariat RNAs and circular RNAs in the pool generated by constitutive and alternative splicing of the dystrophin pre-mRNA. Our results indicate that RNase R treatment can be used to construct an intronic cDNA library, in which majority of the intron lariats are represented. The highly specific activity of RNase R implies its ability to screen for rare intragenic trans-splicing in any target gene with a large background of cis-splicing. Further analysis of the intronic RNA pool from a specific tissue or cell will provide insights into the global profile of alternative splicing.

Regulation of alternative splicing of α‐actinin transcript by Bruno‐like proteins

Background: The Bruno‐like or CELF proteins, such as mammalian CUGBP1 and Etr‐3, Xenopus EDEN‐BP, and Drosophila Bruno (Bru), are regulators of gene expression at the post‐transcriptional level, and contain three RNA‐recognition motifs (RRMs). It has been shown that mammalian CUGBP1 and Etr‐3 regulate alternative splicing of cardiac troponin T pre‐mRNA via binding to CUG‐triplet repeats.

Vegetal localization of the maternal mRNA encoding an EDEN-BP/Bruno-like protein in zebrafish.

Asymmetric distribution of maternal mRNAs has not been well documented in zebrafish. Recently, we have shown that dazl mRNA is localized at the vegetal pole. Here we report a novel zebrafish gene, bruno-like (brul), which provides another example of vegetal mRNA localization. brul encodes an Elav-type RNA-binding protein that belongs to the Bruno-like family that includes mammalian CUG-BP, Xenopus EDEN-BP, and Drosophila Bruno. At 24 hpf, brul mRNA was abundant in lens fiber cells. At the onset of embryogenesis, maternal brul mRNA was detected at the vegetal pole, and it then migrated rapidly toward the blastoderm through yolk cytoplasmic streams. During oogenesis, brul mRNA became localized at the vegetal cortex at stage II, later than dazl mRNA. We found that anchoring of brul mRNA was dependent on microfilaments.

Thr199 phosphorylation targets nucleophosmin to nuclear speckles and represses pre‐mRNA processing

Nucleophosmin (NPM) is a multifunctional phosphoprotein, being involved in ribosome assembly, pre‐ribosomal RNA processing, DNA duplication, nucleocytoplasmic protein trafficking, and centrosome duplication. NPM is phosphorylated by several kinases, including nuclear kinase II, casein kinase 2, Polo‐like kinase 1 and cyclin‐dependent kinases (CDK1 and 2), and these phosphorylations modulate the activity and function of NPM. We have previously identified Thr199 as the major phosphorylation site of NPM mediated by CDK2/cyclin E (and A), and this phosphorylation is involved in the regulation of centrosome duplication. In this study, we further examined the effect of CDK2‐mediated phosphorylation of NPM by using the antibody that specifically recognizes NPM phosphorylated on Thr199. We found that the phospho‐Thr199 NPM localized to dynamic sub‐nuclear structures known as nuclear speckles, which are believed to be the sites of storage and/or assembly of pre‐mRNA splicing factors. Phosphorylation on Thr199 by CDK2/cyclin E (and A) targets NPM to nuclear speckles, and enhances the RNA‐binding activity of NPM. Moreover, phospho‐Thr199 NPM, but not unphosphorylated NPM, effectively represses pre‐mRNA splicing. These findings indicate the involvement of NPM in the regulation of pre‐mRNA processing, and its activity is controlled by CDK2‐mediated phosphorylation on Thr199.

Activation of Pre-mRNA Splicing by Human RNPS1 Is Regulated by CK2 Phosphorylation

ABSTRACT Human RNPS1 was originally characterized as a pre-mRNA splicing activator in vitro and was shown to regulate alternative splicing in vivo. RNPS1 was also identified as a protein component of the splicing-dependent mRNP complex, or exon-exon junction complex (EJC), and a role for RNPS1 in postsplicing processes has been proposed. Here we demonstrate that RNPS1 incorporates into active spliceosomes, enhances the formation of the ATP-dependent A complex, and promotes the generation of both intermediate and final spliced products. RNPS1 is phosphorylated in vivo and interacts with the CK2 (casein kinase II) protein kinase. Serine 53 (Ser-53) of RNPS1 was identified as the major phosphorylation site for CK2 in vitro, and the same site is also phosphorylated in vivo. The phosphorylation status of Ser-53 significantly affects splicing activation in vitro, but it does not perturb the nuclear localization of RNPS1. In vivo experiments indicated that the phosphorylation of RNPS1 at Ser-53 influences the efficiencies of both splicing and translation. We propose that RNPS1 is a splicing regulator whose activator function is controlled in part by CK2 phosphorylation.