前田 明

Abstract mRNA export is an essential pathway for the regulation of gene expression. In humans, closely related RNA helicases, UAP56 and URH49, shape selective mRNA export pathways through the formation of distinct complexes, known as apo-TREX and apo-AREX complexes, and their subsequent remodeling into similar ATP-bound complexes. Therefore, defining the unidentified components of the apo-AREX complex and elucidating the molecular mechanisms underlying the formation of distinct apo-complexes is key to understanding their functional divergence. In this study, we identify additional apo-AREX components physically and functionally associated with URH49. Furthermore, by comparing the structures of UAP56 and URH49 and performing an integrated analysis of their chimeric mutants, we exhibit unique structural features that would contribute to the formation of their respective complexes. This study provides insights into the specific structural and functional diversification of these two helicases that diverged from the common ancestral gene Sub2.

Human pre-mRNA introns vary in size from under fifty to over a million nucleotides. We searched for essential factors involved in the splicing of human short introns by screening siRNAs against 154 human nuclear proteins. The splicing activity was assayed with a model HNRNPH1 pre-mRNA containing short 56-nucleotide intron. We identify a known alternative splicing regulator SPF45 (RBM17) as a constitutive splicing factor that is required to splice out this 56-nt intron. Whole-transcriptome sequencing of SPF45-deficient cells reveals that SPF45 is essential in the efficient splicing of many short introns. To initiate the spliceosome assembly on a short intron with the truncated poly-pyrimidine tract, the U2AF-homology motif (UHM) of SPF45 competes out that of U2AF65 (U2AF2) for binding to the UHM-ligand motif (ULM) of the U2 snRNP protein SF3b155 (SF3B1). We propose that splicing in a distinct subset of human short introns depends on SPF45 but not U2AF heterodimer.

Using TSG101 pre-mRNA, we previously discovered cancer-specific re-splicing of mature mRNA that generates aberrant transcripts/proteins. The fact that mRNA is aberrantly re-spliced in various cancer cells implies there must be an important mechanism to prevent deleterious re-splicing on the spliced mRNA in normal cells. We thus postulated that mRNA re-splicing is controlled by specific repressors, and we searched for repressor candidates by siRNA-based screening for mRNA re-splicing activity. We found that knock-down of EIF4A3, which is a core component of the exon junction complex (EJC), significantly promoted mRNA re-splicing. Remarkably, we could recapitulate cancer-specific mRNA re-splicing in normal cells by knock-down of any of the core EJC proteins, EIF4A3, MAGOH, or RBM8A (Y14), implicating the EJC core as the repressor of mRNA re-splicing often observed in cancer cells. We propose that the EJC core is a critical mRNA quality control factor to prevent over-splicing of mature mRNA.

Pre-mRNA splicing is an essential mechanism for ensuring integrity of the transcriptome in eukaryotes. Therefore, splicing deficiency might cause a decrease in functional proteins and the production of nonfunctional, aberrant proteins. To prevent the production of such aberrant proteins, eukaryotic cells have several mRNA quality control mechanisms. In addition to the known mechanisms, we previously found that transcription elongation is attenuated to prevent the accumulation of pre-mRNA under splicing-deficient conditions. However, the detailed molecular mechanism behind the defect in transcription elongation remains unknown. Here, we showed that the RNA binding protein Rbm38 reduced the transcription elongation defect of the SMEK2 gene caused by splicing deficiency. This reduction was shown to require the N- and C-terminal regions of Rbm38, along with an important role being played by the RNA-recognition motif of Rbm38. These findings advance our understanding of the molecular mechanism of the transcription elongation defect caused by splicing deficiency.

Circular RNAs (circRNAs) are stable non-coding RNAs with a closed circular structure. One of the best studied circRNAs is ciRS-7 (CDR1as), which acts as a regulator of the microRNA miR-7; however, its biosynthetic pathway has remained an enigma. Here we delineate the biosynthetic pathway of ciRS-7. The back-splicing events that form circRNAs are often facilitated by flanking inverted repeats of the primate-specific Alu elements. The ciRS-7 gene lacks these elements, but, instead, we identified a set of flanking inverted elements belonging to the mammalian-wide interspersed repeat (MIR) family. Splicing reporter assays in HEK293 cells demonstrated that these inverted MIRs are required to generate ciRS-7 through back-splicing, and CRISPR/Cas9-mediated deletions confirmed the requirement of the endogenous MIR elements in SH-SY5Y cells. Using bioinformatic searches, we identified several other MIR-dependent circRNAs and confirmed them experimentally. We propose that MIR-mediated RNA circularization is used to generate a subset of mammalian circRNAs.

One of the morphological hallmarks of terminally differentiated secretory cells is highly proliferated membrane of the rough endoplasmic reticulum (ER), but the molecular basis for the high rate of protein biosynthesis in these cells remains poorly documented. An important aspect of ER translational control is the molecular mechanism that supports efficient use of targeted mRNAs in polyribosomes. Here, we identify an enhancement system for ER translation promoted by p180, an integral ER membrane protein we previously reported as an essential factor for the assembly of ER polyribosomes. We provide evidence that association of target mRNAs with p180 is critical for efficient translation, and that SF3b4, an RNA-binding protein in the splicing factor SF3b, functions as a cofactor for p180 at the ER and plays a key role in enhanced translation of secretory proteins. A cis-element in the 5' untranslated region of collagen and fibronectin genes is important to increase translational efficiency in the presence of p180 and SF3b4. These data demonstrate that a unique system comprising a p180-SF3b4-mRNA complex facilitates the selective assembly of polyribosomes on the ER.

TSG101 (Tumor susceptibility 101) gene and its aberrantly spliced isoform, termed TSG101∆154-1054, are tightly linked to tumorigenesis in various cancers. The aberrant TSG101∆154-1054 mRNA is generated from cancer-specific re-splicing of mature TSG101 mRNA. The TSG101∆154-1054 protein protects the full-length TSG101 protein from ubiquitin-mediated degradation, implicating TSG101∆154-1054 protein in the progression of cancer. Here, we confirmed that the presence of TSG101∆154-1054 mRNA indeed caused an accumulation of the TSG101 protein in biopsies of human nasopharyngeal carcinoma (NPC), which was recapitulated by the overexpression of TSG101∆154-1054 in the NPC cell line TW01. We demonstrate the potential function of the TSG101∆154-1054 protein in the malignancy of human NPC with scratch-wound healing and transwell invasion assays. By increasing the stability of the TSG101 protein, TSG101∆154-1054 specifically enhanced TSG101-mediated TW01 cell migration and invasion, suggesting the involvement in NPC metastasis in vivo. This finding sheds light on the functional significance of TSG101∆154-1054 generation via re-splicing of TSG101 mRNA in NPC metastasis and hints at its potential importance as a therapeutic target.

A recent massive parallel sequencing analysis has shown the fact that more than 80% of the human genome is transcribed into RNA. Among many kinds of the non-protein coding RNAs, we focus on the metastasis associated lung adenocarcinoma transcript 1 (MALAT1) that is a long non-coding RNA upregulated in metastatic carcinoma cells. Two molecular functions of MALAT1 have been proposed, one is the control of alternative splicing and the other is the transcriptional regulation. In this review, we document the molecular characteristics and functions of MALAT1 and shed light on the implication in the molecular pathology of various cancers. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.