Biology subjects

Yoshimoto, R.

Publications and source records attributed to Yoshimoto, R..

5 recordsLinked to original sources

bioRxiv2026

Kilohertz electrical stimulation drives widespread neuronal activation and temporal interference imposes focal rhythmic modulation

Temporal interference (TI) stimulation has attracted attention as a non-invasive approach that can target deep brain structures through interacting kilohertz-frequency electric fields. Its basic premise is that TI stimulation evokes neuronal responses through low-frequency amplitude modulation (AM), while the underlying kilohertz-frequency stimulation is ineffective to induce activity, the latter of which has not been tested rigorously at the cellular level. Here, we combined two-photon imaging in mouse models with immunohistochemistry and computational modeling to characterize spatiotemporal patterns of neuronal responses during TI stimulation. We found that kilohertz-frequency stimulation was sufficient to evoke activity without AM. In contrast, AM organized neuronal activity into rhythmic responses at AM frequency, with the strongest entrainment near the center of interference. Thus, kilohertz-frequency stimulation can drive widespread neuronal activation, upon which TI imposes focal rhythmic modulation. Our results provide a framework for refining TI stimulation and highlight the potential of kilohertz-frequency stimulation for brain stimulation.

neuroscience↗

bioRxiv2022

The RBM17/SPF45-SAP30BP interaction is essential for splicing in a subset of human short introns

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 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 NMR analyses demonstrate that a U2AF-homology motif (UHM) in RBM17 binds directly to a newly identified UHM-ligand motif (ULM) in SAP30BP. We show that this RBM17-SAP30BP interaction is required to specifically recruit RBM17 to phosphorylated SF3B1 (SF3b155), a U2 snRNP component in active spliceosomes. We propose a unique mechanism for splicing in a subset of short introns, in which SAP30BP guides RBM17 in the assembly of active spliceosomes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/522300v4_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@12da1c3org.highwire.dtl.DTLVardef@d71e53org.highwire.dtl.DTLVardef@108160corg.highwire.dtl.DTLVardef@1df7275_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefFukumura et al. discover a general splicing mechanism in a subset of human short introns with truncated polypyrimidine tracts. This splicing reaction is mediated by intermediary RBM17-SAP30BP complex, instead of the known U2AF heterodimer. SAP30BP binding to RBM17 may support RBM17 association with active phosphorylated SF3B1 in U2 snRNP. HighlightsO_LIRBM17 (SPF45) is a splicing factor required for a subset of human short introns C_LIO_LISAP30BP is an essential cofactor, which interacts with RBM17 via UHM-ULM binding C_LIO_LIRBM17 forms a weak complex with SAP30BP before its binding with SF3B1 in U2 snRNP C_LIO_LIRBM17-SAP30BP complex supports RBM17 to be recruited to active phosphorylated SF3B1 C_LI

molecular biology↗

bioRxiv2022

A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

Pre-mRNA splicing is an indispensable mechanism for eukaryotic gene expression. Splicing inhibition causes cell cycle arrest at G1 and G2/M phases, which is thought to be one of the reasons for the potent antitumor activity of splicing inhibitors. However, the molecular mechanisms underlying the cell cycle arrest have many unknown aspects. In particular, the mechanism of G2/M-phase arrest caused by splicing inhibition is completely unknown. Here, we found that lower and higher concentrations of pladienolide B caused M-phase and G2-phase arrest, respectively. We analyzed protein levels of cell cycle regulators and found that a truncated form of the p27 CDK inhibitor, named p27*, accumulates in G2-arrested cells. Overexpression of p27* caused partial G2-phase arrest. Conversely, knockdown of p27* accelerated exit from G2/M phase after washout of splicing inhibitor. These results suggest that p27* contributes to G2/M-phase arrest caused by splicing inhibition. We also found that p27* bound to and inhibited M-phase cyclins, although it is well known that p27 regulates G1/S transition. Intriguingly, p27*, but not full-length p27, was resistant to proteasomal degradation and remained in G2/M phase. These results suggest that p27*, which is a very stable truncated protein in G2/M phase, contributes to G2-phase arrest caused by splicing inhibition.

molecular biology↗

bioRxiv2019

A Distinct Subset of Human Short Introns: U2AF Heterodimer Is Replaced by SPF45 (RBM17) as General Splicing Factor

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 identified 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 revealed 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 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.

molecular biology↗

bioRxiv2018

The functional circular RNA, ciRS-7 (CDR1as), is biosynthesized using back-splicing promoted by inverted mammalian-wide MIRs but not primate-specific Alus

Circular RNAs (circRNAs) are stable noncoding RNAs with a closed circular structure. One of the first and best studied circRNAs is ciRS-7 (CDR1as) that acts as a regulator of the microRNA miR-7, however, the biosynthesis pathway has remained an enigma. Here we delineate the biosynthesis pathway of ciRS-7. The back-splicing events that form circRNAs are often facilitated by flanking inverted repeats of the primate-specific Alu elements. 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 a back-splicing and CRISPR/Cas9-mediated deletions confirmed the requirement of the endogenous MIR elements in SH-SY5Y cells. Using bioinformatics searches, we identified several other MIR-dependent circRNAs that we confirmed experimentally. We propose that MIR-mediated RNA circularization constitutes a new widespread biosynthesis principle for mammalian circRNAs.

molecular biology↗