Matrin3 is an RNA-binding protein that is localized in the nuclear matrix. Although various roles in RNA metabolism have been reported for Matrin3, in vivo target RNAs to which Matrin3 binds directly have not been investigated comprehensively so far. Here, we show that Matrin3 binds predominantly to intronic regions of pre-mRNAs. Photoactivatable Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation (PAR-CLIP) analysis using human neuronal cells showed that Matrin3 recognized pyrimidine-rich sequences as binding motifs, including the polypyrimidine tract, a splicing regulatory element. Splicing-sensitive microarray analysis showed that depletion of Matrin3 preferentially increased the inclusion of cassette exons that were adjacent to introns that contained Matrin3-binding sites. We further found that although most of the genes targeted by polypyrimidine tract binding protein 1 (PTBP1) were also bound by Matrin3, Matrin3 could control alternative splicing in a PTBP1-independent manner, at least in part. These findings suggest that Matrin3 is a splicing regulator that targets intronic pyrimidine-rich sequences.

RecJ-like proteins belonging to the DHH family have been proposed to function as oligoribonucleases and 3-phosphoadenosine 5-phosphate (pAp) phosphatases in bacteria and archaea, which do not have Orn (oligoribonuclease) and CysQ (pAp phosphatase) homologs. In this study, we analyzed the biochemical and physiological characterization of the RecJ-like protein TTHA0118 from Thermus thermophilus HB8. TTHA0118 had high enzymatic activity as an oligodeoxyribonucleotide-and oligoribonucleotide-specific exonuclease and as pAp phosphatase. The polarity of degradation was 5 to 3, in contrast to previous reports about Bacillus subtilis NrnA, a RecJ-like protein. TTHA0118 preferentially hydrolyzed short oligodeoxyribonucleotides and oligoribonucleotides, whereas the RecJ exonuclease from T. thermophilus HB8 showed no such length dependence on oligodeoxyribonucleotide substrates. An insertion mutation of the ttha0118 gene led to growth reduction in minimum essential medium. Added 5-mononucleotides, nucleosides, and cysteine increased growth of the ttha0118 mutant in minimum essential medium. The RecJ-like protein Mpn140 from Mycoplasma pneumoniae M129, which cannot synthesize nucleic acid precursors de novo, showed similar biochemical features to TTHA0118. Furthermore, B. subtilis NrnA also hydrolyzed oligo(deoxy)ribonucleotides in a 5-3 direction. These results suggested that these RecJ-like proteins act in recycling short oligonucleotides to mononucleotides and in controlling pAp concentrations in vivo.Nucleases are essential for nucleic acid metabolism and cell viability. DNA repair and recombination require a variety of specific deoxyribonucleases (1, 2). RNA metabolism also requires various ribonucleases (RNases) 2 for a broad range of processes (3, 4). In mRNA turnover, mRNA decay is an important determinant of gene expression and is mediated by several RNases with endonuclease and exonuclease activity. Nucleases belonging to different superfamilies have been identified with endonuclease and exonuclease activities (5-8).In addition, genome sequences have revealed numerous ORFs annotated as putative nucleases.One of the phosphoesterase families is the DHH superfamily (9), which includes phosphodiesterase and exopolyphosphatase (10 -12). RecJ is a DNA exonuclease in the DHH protein superfamily, with DHH motifs I-IV and an associated DHHA1 motif (9), as described in the Pfam database (13). The RecJ protein is a Mg 2ϩ -or Mn 2ϩ -dependent single-stranded DNA (ssDNA)-specific 5Ј-3Ј exonuclease/deoxyribophosphodiesterase and plays a role in homologous recombination, mismatch repair, and base excision repair (14 -19). Although the ORFs annotated as RecJ-related proteins also have DHH and DHHA1 motifs, each RecJ-related protein has to be investigated for nuclease activity similar to RecJ protein. According to the SYSTERS database (20), proteins with DHH and DHHA1 motifs can be further divided into three main families. Representatives of each family are RecJ (N_155469 in SYSTERS), putative poly(A) polymerase (O_142140), and ex...

NanoRNase (Nrn) specifically degrades nucleoside 3′,5′-bisphosphate and the very short RNA, nanoRNA, during the final step of mRNA degradation. The crystal structure of Nrn in complex with a reaction product GMP was determined. The overall structure consists of two domains that are interconnected by a flexible loop and form a cleft. Two Mn2+ ions are coordinated by conserved residues in the DHH motif of the N-terminal domain. GMP binds near the DHHA1 motif region in the C-terminal domain. Our structure enables us to predict the substrate-bound form of Nrn as well as other DHH/DHHA1 phosphoesterase family proteins.

The systematic identification of in vivo targets of nuclear RNA-binding proteins (RBPs) is crucial to elucidate the physiological functions of each RBP. However, it has been difficult to distinguish real targets from nonspecifically bound RNAs and to determine the exact binding sites of each RBP by using a conventional RNA-immunoprecipitation (RIP) method. Photoactivatable Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation (PAR-CLIP) is a recently developed method that relies on RNA-protein cross-linking to reduce the contamination of nonspecifically bound RNAs. Furthermore, in combination with high-throughput sequencing followed by bioinformatic analysis, the exact RBP-binding sites can be identified at a single nucleotide resolution. Here, we describe in detail a PAR-CLIP protocol to prepare cDNA libraries for high-throughput sequencing from RNA fragments that are bound to RBPs not only in the nucleus but also in the cytoplasm.

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