69 research outputs found

    Degradation of Hypomodified tRNA\u3csub\u3ei\u3c/sub\u3e\u3csup\u3eMet\u3c/sup\u3e in vivo Involves RNA-dependent ATPase Activity of the DExH Helicase Mtr4p

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    Effective turnover of many incorrectly processed RNAs in yeast, including hypomodified tRNAi Met, requires the TRAMP complex, which appends a short poly(A) tail to RNA designated for decay. The poly(A) tail stimulates degradation by the exosome. The TRAMP complex contains the poly(A) polymerase Trf4p, the RNA-binding protein Air2p, and the DExH RNA helicase Mtr4p. The role of Mtr4p in RNA degradation processes involving the TRAMP complex has been unclear. Here we show through a genetic analysis that MTR4 is required for degradation but not for polyadenylation of hypomodified tRNAi Met. A suppressor of the trm6-504 mutation in the tRNA m1A58 methyltransferase (Trm6p/Trm61p), which causes a reduced level of tRNAi Met, was mapped to MTR4. This mtr4-20 mutation changed a single amino acid in the conserved helicase motif VI of Mtr4p. The mutation stabilizes hypomodified tRNAi Met in vivo but has no effect on TRAMP complex stability or polyadenylation activity in vivo or in vitro. We further show that purified recombinant Mtr4p displays RNA-dependent ATPase activity and unwinds RNA duplexes with a 3′-to-5′ polarity in an ATP-dependent fashion. Unwinding and RNA-stimulated ATPase activities are strongly reduced in the recombinant mutant Mtr4-20p, suggesting that these activities of Mtr4p are critical for degradation of polyadenylated hypomodified tRNAi Met

    RNA Unwinding by the Trf4/Air2/Mtr4 Polyadenylation (TRAMP) Complex

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    Many RNA-processing events in the cell nucleus involve the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex, which contains the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for processing by the nuclear exosome. In addition, TRAMP functions as an exosome cofactor during RNA degradation, and it has been speculated that this role involves disruption of RNA secondary structure. However, it is unknown whether TRAMP displays RNA unwinding activity. It is also not clear how unwinding would be coordinated with polyadenylation and the function of the RNA helicase Mtr4p in modulating poly(A) addition. Here, we show that TRAMP robustly unwinds RNA duplexes. The unwinding activity of Mtr4p is significantly stimulated by Trf4p/Air2p, but the stimulation of Mtr4p does not depend on ongoing polyadenylation. Nonetheless, polyadenylation enables TRAMP to unwind RNA substrates that it otherwise cannot separate. Moreover, TRAMP displays optimal unwinding activity on substrates with a minimal Mtr4p binding site comprised of adenylates. Our results suggest a model for coordination between unwinding and polyadenylation activities by TRAMP that reveals remarkable synergy between helicase and poly(A) polymerase

    The RNA Helicase Mtr4p Modulates Polyadenylation in the TRAMP Complex

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    SummaryMany steps in nuclear RNA processing, surveillance, and degradation require TRAMP, a complex containing the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for decay or trimming by the nuclear exosome. It has been unclear how polyadenylation by TRAMP differs from polyadenylation by conventional poly(A) polymerase, which produces poly(A) tails that stabilize RNAs. Using reconstituted S. cerevisiae TRAMP, we show that TRAMP inherently suppresses poly(A) addition after only 3–4 adenosines. This poly(A) tail length restriction is controlled by Mtr4p. The helicase detects the number of 3′-terminal adenosines and, over several adenylation steps, elicits precisely tuned adjustments of ATP affinities and rate constants for adenylation and TRAMP dissociation. Our data establish Mtr4p as a critical regulator of polyadenylation by TRAMP and reveal that an RNA helicase can control the activity of another enzyme in a highly complex fashion and in response to features in RNA

    AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation

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    AID/APOBEC family cytosine deaminases, known to function in diverse cellular processes from antibody diversification to mRNA editing, have also been implicated in DNA demethylation, an important process for transcriptional activation. While oxidation-dependent pathways for demethylation have been described, pathways involving deamination of either 5-methylcytosine (mC) or 5-hydroxymethylcytosine (hmC) have emerged as alternatives. Here, we have addressed the biochemical plausibility of deamination-coupled demethylation. We found that purified AID/APOBECs have substantially reduced activity on mC relative to cytosine, their canonical substrate, and no detectable deamination of hmC. This finding was explained by the reactivity of a series of modified substrates, where steric bulk was increasingly detrimental to deamination. Further, upon AID/APOBEC overexpression, the deamination product of hmC was undetectable in genomic DNA, while oxidation intermediates remained detectable. Our results indicate that the steric requirements for cytosine deamination are one intrinsic barrier to the proposed function of deaminases in DNA demethylation

    The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases

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    Reports on bacteria detected in maternal fluids during pregnancy are typically associated with adverse consequences, and whether the female reproductive tract harbours distinct microbial communities beyond the vagina has been a matter of debate. Here we systematically sample the microbiota within the female reproductive tract in 110 women of reproductive age, and examine the nature of colonisation by 16S rRNA gene amplicon sequencing and cultivation. We find distinct microbial communities in cervical canal, uterus, fallopian tubes and peritoneal fluid, differing from that of the vagina. The results reflect a microbiota continuum along the female reproductive tract, indicative of a non-sterile environment. We also identify microbial taxa and potential functions that correlate with the menstrual cycle or are over-represented in subjects with adenomyosis or infertility due to endometriosis. The study provides insight into the nature of the vagino-uterine microbiome, and suggests that surveying the vaginal or cervical microbiota might be useful for detection of common diseases in the upper reproductive tract.Shenzhen Municipal Government of China [JCYJ20160229172757249, JCYJ20150601090833370]; Danish Strategic Research Council [2106-07-0021]; Ole Romer grant from Danish Natural Science Research Council; Solexa project [272-07-0196]SCI(E)ARTICLE
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