研究成果 / Publication

研究成果 / Publication

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The terminal uridylyltransferase, TUT1, builds or repairs the 3′-oligo-uridylylated tail of U6 snRNA. The 3′-oligo-uridylylated tail is the Lsm-binding site for U4/U6 di-snRNP formation and U6 snRNA recycling for pre-mRNA splicing. Here, we report crystallographic and biochemical analyses of human TUT1, which revealed the mechanisms for the specific uridylylation of the 3′-end of U6 snRNA by TUT1. The O2 and O4 atoms of the UTP base form hydrogen bonds with the conserved His and Asn in the catalytic pocket, respectively, and TUT1 preferentially incorporates UMP onto the 3′-end of RNAs. TUT1 recognizes the entire U6 snRNA molecule by its catalytic domains, N-terminal RNA-recognition motifs and a previously unidentified C-terminal RNA-binding domain. Each domain recognizes specific regions within U6 snRNA, and the recognition is coupled with the domain movements and U6 snRNA structural changes. Hence, TUT1 functions as the U6 snRNA-specific terminal uridylyltransferase required for pre-mRNA splicing.

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Human RNA methyltransferase BCDIN3D is overexpressed in breast cancer cells, and is related to the tumorigenic phenotype and poor prognosis of breast cancer. Here, we show that cytoplasmic tRNAHis is the primary target of BCDIN3D in human cells. Recombinant human BCDIN3D, expressed in Escherichia coli, monomethylates the 5΄-monophosphate of cytoplasmic tRNAHis efficiently in vitro. In BCDN3D-knockout cells, established by CRISPR/Cas9 editing, the methyl moiety at the 5΄-monophosphate of cytoplasmic tRNAHis is lost, and the exogenous expression of BCDIN3D in the knockout cells restores the modification in cytoplasmic tRNAHis. BCIDN3D recognizes the 5΄-guanosine nucleoside at position -1 (G-1) and the eight-nucleotide acceptor helix with the G-1-A73 mis-pair at the top of the acceptor stem of cytoplasmic tRNAHis, which are exceptional structural features among cytoplasmic tRNA species. While the monomethylation of the 5΄-monophosphate of cytoplasmic tRNAHis affects neither the overall aminoacylation process in vitro nor the steady-state level of cytoplasmic tRNAHisin vivo, it protects the cytoplasmic tRNAHis transcript from degradation in vitro. Thus, BCDIN3D acts as a cytoplasmic tRNAHis-specific 5΄-methylphosphate capping enzyme. The present results also suggest the possible involvement of the monomethylation of the 5΄-monophosphate of cytoplasmic tRNAHis and/or cytoplasmic tRNAHis itself in the tumorigenesis of breast cancer cells.

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The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/ N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genomewide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ~11 bp.

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  • Authors Linyi Gao, David B T Cox, Winston X Yan, John C Manteiga, Martin W Schneider, Takashi Yamano, Hiroshi Nishimasu, Osamu Nureki, Nicola Crosetto, *Feng Zhang

The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1.

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  • Authors +*Hiroshi Nishimasu, +Takashi Yamano, Linyi Gao, Feng Zhang, Ryuichiro Ishitani, *Osamu Nureki

NEAT1_2 long noncoding RNA (lncRNA) is the molecular scaffold of paraspeckle nuclear bodies. Here, we report an improved RNA extraction method: extensive needle shearing or heating of cell lysate in RNA extraction reagent improved NEAT1_2 extraction by 20‐fold (a property we term “semi‐extractability”), whereas using a conventional method NEAT1_2 was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50 NEAT1_2 molecules are present in a single paraspeckle. Another architectural lncRNA, IGS16, also exhibited similar semi‐extractability. A comparison of RNA‐seq data from needle‐sheared and control samples revealed the existence of multiple semi‐extractable RNAs, many of which were localized in subnuclear granule‐like structures. The semi‐extractability of NEAT1_2 correlated with its association with paraspeckle proteins and required the prion‐like domain of the RNA‐binding protein FUS. This observation suggests that tenacious RNA–protein and protein–protein interactions, which drive nuclear body formation, are responsible for semi‐extractability. Our findings provide a foundation for the discovery of the architectural RNAs that constitute nuclear bodies.

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miRNAs play critical roles in various biological processes by targeting specific mRNAs. Current approaches to identifying miRNA targets are insufficient for elucidation of a miRNA regulatory network. Here, we created a cell-based screening system using a luciferase reporter library composed of 4,891 full-length cDNAs, each of which was integrated into the 3' UTR of a luciferase gene. Using this reporter library system, we conducted a screening for targets of miR-34a, a tumor-suppressor miRNA. We identified both previously characterized and previously uncharacterized targets. miR-34a overexpression in MDA-MB-231 breast cancer cells repressed the expression of these previously unrecognized targets. Among these targets, GFRA3 is crucial for MDA-MB-231 cell growth, and its expression correlated with the overall survival of patients with breast cancer. Furthermore, GFRA3 was found to be directly regulated by miR-34a via its coding region. These data show that this system is useful for elucidating miRNA functions and networks.

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It remains unclear how post-transcriptional gene silencing (PTGS) in plants discriminates aberrant RNAs from canonical messenger RNAs (mRNAs). The key step of plant PTGS is the conversion of aberrant RNAs into double-stranded RNAs by RNA-DEPENDENT RNA POLYMERASE6 (RDR6). Here, we show that RDR6 itself selects aberrant poly(A)-less mRNAs over canonical polyadenylated mRNAs as templates at the initiation step of complementary strand synthesis. This mechanism can be viewed as an innate safeguard against 'self-attack' by PTGS.

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The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5′-NNNVRYM-3′ as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering.

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  • Authors +Yamada M, +Watanabe Y, Gootenberg JS, Hirano H, Ran FA, Nakane T, Ishitani R, Zhang F, *Nishimasu H, *Nureki O

PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs) and silence transposable elements in animal gonads. Here, we report the crystal structure of a silkworm PIWI-clade Argonaute, Siwi, bound to the endogenous piRNA, at 2.4 Å resolution. Siwi adopts a bilobed architecture consisting of N-PAZ and MID-PIWI lobes, in which the 5' and 3' ends of the bound piRNA are anchored by the MID-PIWI and PAZ domains, respectively. A structural comparison of Siwi with AGO-clade Argonautes reveals notable differences in their nucleic-acid-binding channels, likely reflecting the distinct lengths of their guide RNAs and their mechanistic differences in guide RNA loading and cleavage product release. In addition, the structure reveals that Siwi and prokaryotic, but not eukaryotic, AGO-clade Argonautes share unexpected similarities, such as metal-dependent 5'-phosphate recognition and a potential structural transition during the catalytic-tetrad formation. Overall, this study provides a critical starting point toward a mechanistic understanding of piRNA-mediated transposon silencing.

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Paraspeckles are nuclear bodies built on the long noncoding RNA Neat1, which regulates a variety of physiological processes including cancer progression and corpus luteum formation. To obtain further insight into the molecular basis of the function of paraspeckles, we performed fine structural analyses of these nuclear bodies using structural illumination microscopy. Notably, paraspeckle proteins are found within different layers along the radially arranged bundles of Neat1 transcripts, forming a characteristic core-shell spheroidal structure. In cells lacking the RNA binding protein Fus, paraspeckle spheroids are disassembled into smaller particles containing Neat1, which are diffusely distributed in the nucleoplasm. Sequencing analysis of RNAs purified from paraspeckles revealed that AG-rich transcripts associate with Neat1, which are distributed along the shell of the paraspeckle spheroids. We propose that paraspeckles sequester core components inside the spheroids, whereas the outer surface associates with other components in the nucleoplasm to fulfill their function.

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