Takashi Miki

XRN2 is an evolutionarily conserved 5’-to-3’ exoribonuclease, which degrades or trims various types of RNA in the nucleus. Although XRN-2 is essential for embryogenesis, larval development and reproduction in Caenorhabditis elegans, relevant molecular pathways remain unidentified. Here we create a germline-specific xrn-2 conditional mutant and perform a mutagenesis screen for suppressors of sterility. Loss-of-function alleles of dpy-10, osr-1, ptr-6 and C34C12.2 genes are identified. Depletion of DPY-10, OSR-1 or PTR-6 increases expression of gpdh-1 that encodes a glycerol-3-phosphate dehydrogenase, thereby elevates glycerol accumulation to suppress sterility of the mutant. The C34C12.2 protein is predominantly localized in the nucleolus of germ cells and shows a similarity to Saccharomyces cerevisiae Net1, which is involved in rDNA silencing. Depletion of NRDE-2, a putative interacting partner of C34C12.2 and a component of the nuclear RNAi machinery, restores fertility to the xrn-2 conditional mutant. These results may help to identify an essential role of XRN-2 in germline development.

XRN2 is a 5’-to-3’ exoribonuclease that is predominantly localized in the nucleus. By degrading or trimming various classes of RNA, XRN2 contributes to essential processes in gene expression such as transcription termination and ribosome biogenesis. Despite limited substrate specificity in vitro, XRN2 targets a specific subset of RNA by interacting with other proteins in cells. Here we review the functions of proteins that have an evolutionarily conserved XRN2-binding domain, XTBD. These proteins modulate activity of XRN2 by stabilizing it, controlling its subcellular localization or recruiting it to specific RNA targets, and thereby impact on various cellular processes. Key words: RNA regulation, XRN2, XTBD, ribosome biogenesis, subcellular localization

Transcription termination determines the ends of transcriptional units and thereby ensures the integrity of the transcriptome and faithful gene regulation. Studies in yeast and human cells have identified the exoribonuclease XRN2 as a key termination factor for protein-coding genes. Here we performed a genome-wide investigation of RNA polymerase II (Pol II) transcription termination in XRN2-deficient Caenorhabditis elegans and observed two distinct modes of termination. Although a subset of genes requires XRN2, termination of other genes appears both independent of, and refractory to, XRN2. XRN2 independence is not merely a consequence of failure to recruit XRN2, since XRN2 is present on—and promotes Pol II accumulation near the polyadenylation sites of—both gene classes. Unexpectedly, promoters instruct the choice of termination mode, but XRN2-independent termination additionally requires a compatible region downstream from the 3′ end cleavage site. Hence, different termination mechanisms may work with different configurations of Pol II complexes dictated by promoters.

XRN2 is a conserved 5’→3’ exoribonuclease that complexes with proteins that contain XRN2-binding domains (XTBDs). In Caenorhabditis elegans (C. elegans), the XTBD-protein PAXT-1 stabilizes XRN2 to retain its activity. XRN2 activity is also promoted by 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1) through hydrolysis of an endogenous XRN inhibitor 3’-phosphoadenosine-5'-phosphate (PAP). Here, we find through unbiased screening that loss of bpnt-1 function suppresses lethality caused by paxt-1 deletion. This unexpected finding is explained by XRN2 autoregulation, which occurs through repression of a cryptic promoter activity and destabilization of the xrn-2 transcript. De-repression appears to be triggered such that more robust XRN2 perturbation, by elimination of both PAXT-1 and BPNT1, is less detrimental to worm viability than absence of PAXT-1 alone. Indeed, we find that two distinct XRN2 repression mechanisms are alleviated at different thresholds of XRN2 inactivation. Like more than 15% of C. elegans genes, xrn-2 occurs in an operon, and we identify additional operons under its control, consistent with a broader function of XRN2 in polycistronic gene regulation. Regulation occurs through intercistronic regions that link genes in an operon, but a part of the mechanisms may allow XRN2 to operate on monocistronic genes in organisms lacking operons. XRN2 is a conserved eukaryotic protein that controls gene expression by degrading or processing various types of RNA. Here we find that XRN2 negatively regulates its own levels in the nematode C. elegans. In response to reduction of XRN2 activity, this self-repression is alleviated, increasing xrn-2 mRNA and thus protein production, which restores robust XRN2 activity. Although XRN2 and its upstream gene are transcribed from a single promoter as a gene expression unit called “operon”, XRN2 regulates only itself. It does so by inactivating a cryptic promoter that exists between the two genes and by destabilizing its own nascent transcript. Many other C. elegans genes (>15%) occur in operons, and we identify additional operons that XRN2 regulates through an analogous mechanism. Thus we find a novel function of XRN2 in modulating expression of genes in operons including itself. As one of the mechanisms could operate on genes outside operons, XRN2 may also regulate gene expression in organisms lacking operonic gene organization.

The spliceosome is a large molecular machine that serves to remove the intervening sequences that are present in most eukaryotic pre-mRNAs. At its core are five small nuclear ribonucleoprotein complexes, the U1, U2, U4, U5 and U6 snRNPs, which undergo dynamic rearrangements during splicing. Their reutilization for subsequent rounds of splicing requires reversion to their original configurations, but little is known about this process. Here, we show that ZK863.4/USIP-1 (U Six snRNA-Interacting Protein-1) is a ribonucleotidyl transferase that promotes accumulation of the Caenorhabditis elegans U6 snRNA. Endogenous USIP-1–U6 snRNA complexes lack the Lsm proteins that constitute the protein core of the U6 snRNP, but contain the U6 snRNP recycling factor SART3/B0035.12. Furthermore, co-immunoprecipitation experiments suggest that SART3 but not USIP-1 occurs also in a separate complex containing both the U4 and U6 snRNPs. Based on this evidence, genetic interaction between usip-1 and sart-3, and the apparent dissociation of Lsm proteins from the U6 snRNA during spliceosome activation, we propose that USIP-1 functions upstream of SART3 to promote U6 snRNA recycling.

Although XRN2 proteins are highly conserved eukaryotic 5′→3′ exonucleases, little is known about their function in animals. Here, we characterize Caenorhabditis elegans XRN2, which we find to be a broadly and constitutively expressed nuclear protein. An xrn-2 null mutation or loss of XRN2 catalytic activity causes a molting defect and early larval arrest. However, by generating a conditionally mutant xrn-2ts strain de novo through an approach that may be also applicable to other genes of interest, we reveal further functions in fertility, during embryogenesis and during additional larval stages. Consistent with the known role of XRN2 in controlling microRNA (miRNA) levels, we can demonstrate that loss of XRN2 activity stabilizes some rapidly decaying miRNAs. Surprisingly, however, other miRNAs continue to decay rapidly in xrn-2ts animals. Thus, XRN2 has unanticipated miRNA specificity in vivo, and its diverse developmental functions may relate to distinct substrates. Finally, our global analysis of miRNA stability during larval stage 1 reveals that miRNA passenger strands (miR*s) are substantially less stable than guide strands (miRs), supporting the notion that the former are mostly byproducts of biogenesis rather than a less abundant functional species.

Different classes of RNA function in various cellular processes, and their biogenesis and turnover involve diverse RNases for processing and degradation. XRN2 is a 5′→3′ exoribonuclease that is evolutionarily conserved in eukaryotes. It is predominantly localized in the nucleus and recognizes single-stranded RNA with a 5′-terminal monophosphate to degrade it processively to mononucleotides. In the present paper, we review functions of XRN2 and its cofactors in maturation, surveillance and activity control of several classes of RNA such as pre-mRNA (precursor mRNA), rRNA and snoRNA (small nucleolar RNA).

Staufen2 (Stau2), a double-stranded RNA-binding protein, is a component of neuronal RNA granules, which are dendritic mRNA transport machines. Although Stau2 is thought to be involved in the dendritic targeting of several mRNAs in neurons, the mechanism whereby Stau2 regulates these mRNAs is unknown. To elucidate the functions of Stau2, we screened for novel binding partners by affinity purification of GST-tagged Stau2 from 293F cells. Three RNA helicases, RNA helicase A, Upf1 and Mov10, were identified in Stau2-containing complexes. We focused our studies on Upf1, a key player in nonsense-mediated mRNA decay. Stau2 was found to bind directly to Upf1 in an RNA-independent manner in vitro. Tethering Stau2 to the 3'-untranslated region (UTR) of a reporter gene had little effect on its expression in HeLa cells. In contrast, when the same tethering assay was performed in 293F cells, we observed an increase in reporter protein levels. This upregulation of protein expression by Stau2 turned out to be dependent on Upf1. Moreover, we found that in 293F cells, Stau2 upregulates the reporter mRNA level in an Upf1-independent manner. These results indicate that the recruitment of Stau2 alone or in combination with Upf1 differentially affects the fate of mRNAs. Moreover, the results suggest that Stau2-mediated fate determination could be executed in a cell type-specific manner.

Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. Although Rho is critically involved in cytokinesis, it remains elusive how Rho effectors and other regulators of cytoskeletons work together to accomplish this process. Here we focused on mDia2, an mDia isoform involved in cytokinesis of NIH 3T3 cells, and analyzed mechanisms of its localization in cytokinesis. We found that targeting of mDia2 to the cleavage furrow requires not only its binding to RhoA but also its diaphanous-inhibitory domain (DID). We then performed pulldown assays using a fragment containing the latter domain as a bait and identified anillin as a novel mDia2 interaction partner. The anillin-binding is competitive with the diaphanous autoregulatory domain (DAD) of mDia2 in its autoinhibitory interaction. A series of RNA interference and functional rescue experiments has revealed that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.