研究成果 / Publication

研究成果 / Publication

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Cpf1 is an RNA-guided endonuclease of a type V CRISPR-Cas system that has been recently harnessed for genome editing. Here, we report the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with the guide RNA and its target DNA at 2.8 Å resolution. AsCpf1 adopts a bilobed architecture, with the RNA-DNA heteroduplex bound inside the central channel. The structural comparison of AsCpf1 with Cas9, a type II CRISPR-Cas nuclease, reveals both striking similarity and major differences, thereby explaining their distinct functionalities. AsCpf1 contains the RuvC domain and a putative novel nuclease domain, which are responsible for cleaving the non-target and target strands, respectively, and for jointly generating staggered DNA double-strand breaks. AsCpf1 recognizes the 5'-TTTN-3' protospacer adjacent motif by base and shape readout mechanisms. Our findings provide mechanistic insights into RNA-guided DNA cleavage by Cpf1 and establish a framework for rational engineering of the CRISPR-Cpf1 toolbox.

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The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9.

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Alternative splicing of pre-mRNAs can regulate expression of protein-coding genes by generating unproductive mRNAs rapidly degraded by nonsense-mediated mRNA decay (NMD). Many of the genes directly regulated by alternative splicing coupled with NMD (AS-NMD) are related to RNA metabolism, but the repertoire of genes regulated by AS-NMD in vivo is to be determined. Here, we analyzed transcriptome data of wild-type and NMD-defective mutant strains of the nematode worm Caenorhabditis elegans and demonstrate that eight of the 82 cytoplasmic ribosomal protein (rp) genes generate unproductively spliced mRNAs. Knockdown of any of the eight rp genes exerted a dynamic and compensatory effect on alternative splicing of its own transcript and inverse effects on that of the other rp genes. A large subunit protein L10a, termed RPL-1 in nematodes, directly and specifically binds to an evolutionarily conserved 39-nt stretch termed L10ARE between the two alternative 5′ splice sites in its own pre-mRNA to switch the splice site choice. Furthermore, L10ARE-mediated splicing autoregulation of the L10a-coding gene is conserved in vertebrates. These results indicate that L10a is an evolutionarily conserved splicing regulator and that homeostasis of a subset of the rp genes are regulated at the level of pre-mRNA splicing in vivo.

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PIWI-interacting RNAs (piRNAs) play a crucial role in transposon silencing in animal germ cells. In piRNA biogenesis, single-stranded piRNA intermediates are loaded into PIWI-clade proteins and cleaved by Zucchini/MitoPLD, yielding precursor piRNAs (pre-piRNAs). Pre-piRNAs that are longer than the mature piRNA length are then trimmed at their 3′ ends. Although recent studies implicated the Tudor domain protein Papi/Tdrkh in pre-piRNA trimming, the identity of Trimmer and its relationship with Papi/Tdrkh remain unknown. Here, we identified PNLDC1, an uncharacterized 3′-5′ exonuclease, as Trimmer in silkworms. Trimmer is enriched in the mitochondrial fraction and binds to Papi/Tdrkh. Depletion of Trimmer and Papi/Tdrkh additively inhibits trimming, causing accumulation of ∼35–40-nt pre-piRNAs that are impaired for target cleavage and prone to degradation. Our results highlight the cooperative action of Trimmer and Papi/Tdrkh in piRNA maturation.

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The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox.

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Many long noncoding RNAs (lncRNAs) are reported to be dysregulated in human cancers and play critical roles in tumor development and progression. Furthermore, it has been reported that many lncRNAs regulate gene expression by recruiting chromatin remodeling complexes to specific genomic loci or by controlling transcriptional or posttranscriptional processes. Here we show that an lncRNA termed UPAT [ubiquitin-like plant homeodomain (PHD) and really interesting new gene (RING) finger domain-containing protein 1 (UHRF1) Protein Associated Transcript] is required for the survival and tumorigenicity of colorectal cancer cells. UPAT interacts with and stabilizes the epigenetic factor UHRF1 by interfering with its β-transducin repeat-containing protein (TrCP)-mediated ubiquitination. Furthermore, we demonstrate that UHRF1 up-regulates Stearoyl-CoA desaturase 1 and Sprouty 4, which are required for the survival of colon tumor cells. Our study provides evidence for an lncRNA that regulates protein ubiquitination and degradation and thereby plays a critical role in the survival and tumorigenicity of tumor cells. Our results suggest that UPAT and UHRF1 may be promising molecular targets for the therapy of colon cancer.

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This special issue aims to assemble available knowledge on long noncoding RNAs (lncRNAs) and provide future research directions for discovering the molecular functions of this emerging family of molecules. The genomes of eukaryotes, particularly mammalian species including human and mouse, possess large chunks of nonprotein-coding regions. Only 2% of the human genome is dedicated to coding for proteins; the remainder is constituted of noncoding regions, which are for the most part functionally unannotated. At the beginning of the postgenomic era, transcriptome genome-wide analyses in various organisms unexpectedly revealed that large portions of the mammalian genome produce numerous transcripts that lack protein-coding potential. Among these RNAs, noncoding transcripts longer than 200 nt are arbitrary referred to as “lncRNAs”. Many lncRNAs are expressed at low levels, exhibit tissue- or cell type specific expression patterns, and are not as well conserved between species as protein-coding mRNAs. LncRNAs share common features with protein-coding mRNAs; for instance, with few exceptions, they are transcribed by RNA polymerase II, possess the canonical cap structure at their 5′ termini, and their 3′ termini are polyadenylated. Nevertheless, many lncRNAs are not subject to nuclear export and function within the nucleus, which is in sharp contrast to mRNAs that are transported to the cytoplasm and translated into proteins. Notably, a group of lncRNAs, once classified as lncRNAs, have now been found to encode small polypeptides, making it necessary to establish new methods to distinguish lncRNAs from polypeptide-coding RNAs...

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A number of non-membranous cellular bodies have been identified in higher eukaryotes, and these bodies contain a specific set of proteins and RNAs that are used to fulfill their functions. The size of these RNA-containing cellular bodies is usually on a submicron scale, making it difficult to observe fine structures using optical microscopy due to the diffraction limitation of visible light. Recently, microscope companies have released super-resolution microscopes that were developed using different principles, enabling the observation of sub-micron structures not resolvable in conventional fluorescent microscopy. Here, we describe multi-color fluorescent in situ hybridization techniques optimized for the simultaneous detection of RNA and proteins using super-resolution microscopy, namely structured illumination microscopy (SIM).

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Short interspersed elements (SINEs) comprise a significant portion of mammalian genomes and regulate gene expression through a variety of mechanisms. Here, we show that Myodonta clade-specific 4.5S RNAH (4.5SH), an abundant nuclear noncoding RNA that is highly homologous to the retrotransposon SINE B1, controls the expression of reporter gene that contains the antisense insertion of SINE B1 via nuclear retention. The depletion of endogenous 4.5SH with antisense oligonucleotides neutralizes the nuclear retention and changes the subcellular distribution of the reporter transcripts containing the antisense SINE B1 insertion. Importantly, endogenous transcripts with antisense SINE B1 were increased in the cytoplasm after knockdown of 4.5SH, leading to a decrease in cellular growth. We propose a tentative hypothesis that the amplification of the 4.5SH cluster in specific rodent species might delineate their evolutionary direction via the regulation of genes containing the antisense insertion of SINE B1.

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Piwi-interacting RNAs (piRNAs) suppress transposon activity in animal germ cells. In the Drosophila ovary, primary Aubergine (Aub)-bound antisense piRNAs initiate the ping-pong cycle to produce secondary AGO3-bound sense piRNAs. This increases the number of secondary Aub-bound antisense piRNAs that can act to destroy transposon mRNAs. Here we show that Krimper (Krimp), a Tudor-domain protein, directly interacts with piRNA-free AGO3 to promote symmetrical dimethylarginine (sDMA) modification, ensuring sense piRNA-loading onto sDMA-modified AGO3. In aub mutant ovaries, AGO3 associates with ping-pong signature piRNAs, suggesting AGO3's compatibility with primary piRNA loading. Krimp sequesters ectopically expressed AGO3 within Krimp bodies in cultured ovarian somatic cells (OSCs), in which only the primary piRNA pathway operates. Upon krimp-RNAi in OSCs, AGO3 loads with piRNAs, further showing the capacity of AGO3 for primary piRNA loading. We propose that Krimp enforces an antisense bias on piRNA pools by binding AGO3 and blocking its access to primary piRNAs.

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