Kazuhiro FUKUMURA

Human pre-mRNA splicing requires the removal of introns with highly variable lengths, from tens to over a million nucleotides. Therefore, mechanisms of intron recognition and splicing are likely not universal. Recently, we reported that splicing in a subset of human short introns with truncated polypyrimidine tracts depends on RBM17 (SPF45), instead of the canonical splicing factor U2 auxiliary factor (U2AF) heterodimer. Here, we demonstrate that SAP30BP, a factor previously implicated in transcriptional control, is an essential splicing cofactor for RBM17. In vitro binding and nuclear magnetic resonance analyses demonstrate that a U2AF-homology motif (UHM) in RBM17 binds directly to a newly identified UHM-ligand motif in SAP30BP. We show that this RBM17-SAP30BP interaction is required to specifically recruit RBM17 to phosphorylated SF3B1 (SF3b155), a U2 small nuclear ribonucleoprotein (U2 snRNP) component in active spliceosomes. We propose a mechanism for splicing in a subset of short introns, in which SAP30BP guides RBM17 in the assembly of active spliceosomes.

BACKGROUND/AIM: Immune checkpoint inhibitors (ICIs) are currently a standard treatment tool for non-small cell lung cancer (NSCLC). RNA-binding motif protein 17 (RBM17), a splicing factor, is frequently over-expressed in NSCLC, but little is known about the role of RBM17 in the efficacy of ICIs for NSCLC. Thus, we investigated the correlation between RBM17 expression and ICI efficacy in NSCLC. PATIENTS AND METHODS: Biopsy or surgical specimens were collected from patients with advanced or recurrent NSCLC who received ICI monotherapy or chemo-immunotherapy in a first-line setting. RBM17 expression was examined using immunohistochemistry. The correlation between the efficacy of ICI monotherapy or chemo-immunotherapy and RBM17 expression was evaluated. RESULTS: Among the 218 cases, 115 (52.8%) cases were positive for RBM17 expression. RBM17 expression was not associated with the objective response rate (ORR) or progression-free survival (PFS) in either of the ICI monotherapy or chemo-immunotherapy groups. However, among those with a low PD-L1 expression level (PD-L1 <50%; n=86), RBM17 expression was significantly associated with a better ORR (p=0.045) and a better PFS (p<0.001) in the ICI monotherapy group, and was significantly associated with a poor ORR in the chemo-immunotherapy group (p=0.041). CONCLUSION: RBM17 might be a useful predictive marker for a higher efficacy of ICI monotherapy in NSCLC patients with a low PD-L1 expression level.

Laminins are heterotrimeric ECM proteins composed of α, β, and γ chains. The γ2 chain (Lm-γ2) is a frequently expressed monomer and its expression is closely associated with cancer progression. Laminin-γ2 contains an epidermal growth factor (EGF)-like domain in its domain III (DIII or LEb). Matrix metalloproteinases can cleave off the DIII region of Lm-γ2 that retains the ligand activity for EGF receptor (EGFR). Herein, we show that a novel short form of Lm-γ2 (Lm-γ2F) containing DIII is generated without requiring MMPs and chromosomal translocation between LAMC2 on chromosome 1 and NR6A1 gene locus on chromosome 9 in human ovarian cancer SKOV3 cells. Laminin-γ2F is expressed as a truncated form lacking domains I and II, which are essential for its association with Lm-α3 and -β3 chains of Lm-332. Secreted Lm-γ2F can act as an EGFR ligand activating the EGFR/AKT pathways more effectively than does the Lm-γ2 chain, which in turn promotes proliferation, survival, and motility of ovarian cancer cells. LAMC2-NR6A1 translocation was detected using in situ hybridization, and fusion transcripts were expressed in ovarian cancer cell tissues. Overexpression and suppression of fusion transcripts significantly increased and decreased the tumorigenic growth of cells in mouse models, respectively. To the best of our knowledge, this is the first report regarding a fusion gene of ECM showing that translocation of LAMC2 plays a crucial role in the malignant growth and progression of ovarian cancer cells and that the consequent product is a promising therapeutic target against ovarian cancers.

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.

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.