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Uridylation-dependent RNA decay is a widespread eukaryotic pathway modulating RNA homeostasis. Terminal uridylyltransferases (Tutases) add untemplated uridyl residues to RNA 3′-ends, marking them for degradation by the U-specific exonuclease Dis3L2. In Schizosaccharomyces pombe , Cid1 uridylates a variety of RNAs. In this study, we investigate the prevalence and impact of uridylation-dependent RNA decay in S. pombe by transcriptionally profiling cid1 and dis3L2 deletion strains. We found that the exonuclease Dis3L2 represents a bottleneck in uridylation-dependent mRNA decay, whereas Cid1 plays a redundant role that can be complemented by other Tutases. Deletion of dis3L2 elicits a cellular stress response, upregulating transcription of genes involved in protein folding and degradation. Misfolded proteins accumulate in both deletion strains, yet only trigger a strong stress response in dis3L2 deficient cells. While a deletion of cid1 increases sensitivity to protein misfolding stress, a dis3L2 deletion showed no increased sensitivity or was even protective. We furthermore show that uridylyl- and adenylyltransferases cooperate to generate a 5′-N x AUUAAAA-3′ RNA motif on dak2 mRNA. Our studies elucidate the role of uridylation-dependent RNA decay as part of a global mRNA surveillance, and we found that perturbation of this pathway leads to the accumulation of misfolded proteins and elicits cellular stress responses.

tRNA guanylyltransferase (Thg1) has unique reverse (3'-5') polymerase activity occurring in all three domains of life. Most eukaryotic Thg1 homologs are essential genes involved in tRNA maturation. These enzymes normally catalyze a single 5' guanylation of tRNA lacking the essential G identity element required for aminoacylation. Recent studies suggest that archaeal type Thg1, which includes most archaeal and bacterial Thg1 enzymes is phylogenetically distant from eukaryotic Thg1. Thg1 is evolutionarily related to canonical 5'-3' forward polymerases but catalyzes reverse 3'-5'polymerization. Similar to its forward polymerase counterparts, Thg1 encodes the conserved catalytic palm domain and fingers domain. Here we investigate the minimal requirements for reverse polymerization. We show that the naturally occurring minimal Thg1 enzyme from Ignicoccus hospitalis (IhThg1), which lacks parts of the conserved fingers domain, is catalytically active. And adds all four natural nucleotides to RNA substrates, we further show that the entire fingers domain of Methanosarcina acetivorans Thg1 and Pyrobaculum aerophilum Thg1 (PaThg1) is dispensable for enzymatic activity. In addition, we identified residues in yeast Thg1 that play a part in preventing extended polymerization. Mutation of these residues with alanine resulted in extended reverse polymerization. PaThg1 was found to catalyze extended, template dependent tRNA repair, adding up to 13 nucleotides to a truncated tRNA substrate. Sequencing results suggest that PaThg1 fully restored the near correct sequence of the D- and acceptor stem, but also produced incompletely and incorrectly repaired tRNA products. This research forms the basis for future engineering efforts towards a high fidelity, template dependent reverse polymerase.

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Introduction The fabellofibular ligament (FFL) is a component of the posterolateral corner (PLC) of the knee and is an anatomically variable static stabilizer. Several investigations have reported prevalence rates for the FFL among their subjects, but no overall prevalence rate has been reported. Materials and Methods We conducted a meta‐analysis of all relevant studies reporting prevalence rates of the FFL according to PRISMA and AQUA guidelines and pooled prevalence data using MetaXL 5.3. We also conducted a retroactive magnetic resonance imaging (MRI) study of 100 knees to assess FFL and fabella prevalence. Results Twenty‐one studies (from 18 articles) and our MRI data were used in this meta‐analysis (n = 1,176 knees). The pooled prevalence estimate (PPE) for FFL absence was 37.4% (95% confidence interval [CI], 24.5–51.3%). When divided by continent, PPEs of FFL absence were 31.5% (95% CI, 1.4–72.7%), 58.2% (95% CI, 44.1–71.6%), and 29.0% (95% CI, 14.7–45.7%) for North American, European, and Asian subjects, respectively. Cadaveric and MRI studies showed PPEs of FFL absence of 31.5% and 49.7%, respectively. Our MRI results showed PPEs of FFL and fabella absences of 42.0 and 80.0%, respectively. Conclusions Understanding prevalence rates and anatomical geometry of the FFL will assist surgeons in repairing PLC injuries. Our MRI data and previous studies suggest the FFL is frequently present in knees lacking a fabella. Based on the observations of this study, we propose the ligament be renamed the gastrocnemiofibular ligament.