A Comparative Study of the Structural Dynamics of Four Terminal Uridylyl Transferases.

African trypanosomiasis occurs in 36 countries in sub-Saharan Africa with 10,000 reported cases annually. No definitive remedy is currently available and if left untreated, the disease becomes fatal. Structural and biochemical studies of trypanosomal terminal uridylyl transferases (TUTases) demonstrated their functional role in extensive uridylate insertion/deletion of RNA. Trypanosoma brucei RNA Editing TUTase 1 (TbRET1) is involved in guide RNA 3' end uridylation and maturation, while TbRET2 is responsible for U-insertion at RNA editing sites. Two additional TUTases called TbMEAT1 and TbTUT4 have also been reported to share similar function. TbRET1 and TbRET2 are essential enzymes for the parasite viability making them potential drug targets. For this study, we clustered molecular dynamics (MD) trajectories of four TUTases based on active site shape measured by Pocket Volume Measurer (POVME) program. Among the four TUTases, TbRET1 exhibited the largest average pocket volume, while TbMEAT1's and TbTUT4's active sites displayed the most flexibility. A side pocket was also identified within the active site in all TUTases with TbRET1 having the most pronounced. Our results indicate that TbRET1's larger side pocket can be exploited to achieve selective inhibitor design as FTMap identifies it as a druggable pocket

Anatomy of the disease-related Dis3L2 ribonuclease: dissecting the amino acids responsible for substrate specificity

Ribonucleases are enzymes which perform the processing and degradation of all types of RNA, being critical for the tight post-transcriptional regulation of gene expression. Three homologues from the RNase II/RNB family of exoribonucleases are found in eukaryotes: Dis3, Dis3L1, and Dis3L2. The first two associate with the RNA exosome, while Dis3L2 participates in an alternative degradation pathway, depending on 3’-uridylation by terminal uridylyl transferases and poly(U) polymerases. The first insights on protein–RNA interactions were obtained analysing the mouse Dis3L2–oligo(U) structure, supporting the distinctive preference of Dis3L2 for uridylated substrates. To date, Dis3L2 has already been associated with cellular processes as relevant as RNA surveillance, cell proliferation and differentiation. Human DIS3L2 defects have been related to several cancers, alongside with Perlman Syndrome, a rare foetal overgrowth disorder, consequently increasing the risk for Wilms Tumour, a malignant kidney tumour commonly affecting children. Nevertheless, much remains unknown about the mechanism of action of Dis3L2 and its peculiar substrate preference. In this work, fission yeast was used as an eukaryotic microorganism model for studying a considerably important ribonuclease in human diseases. Our aim was the identification of particular amino acids potentially crucial for activity and substrate specificity of Schizosaccharomyces pombe Dis3L2 (SpDis3L2), namely those involved in its preference for uridylated substrates. Thus, wild-type and point mutants of SpDis3L2 were overexpressed and purified. A thermal shift assay allowed the improvement of protein stability and enhanced full-length protein purification. Subsequently, the exoribonucleolytic activity of SpDis3L2 versions was analysed over different RNA substrates, using an inactive mutant as control. We uncovered that all tested mutants were active, with several of them differing from the wild-type regarding their level of activity and their preference for uridylated substrates. Therefore, these results pave the way for the analysis of other protein versions with combinations of these point mutations.As ribonucleases são enzimas responsáveis pelo processamento e degradação do RNA, sendo essenciais na regulação pós-transcricional da expressão génica. Em eucariotas, encontramos três homólogos das exorribonucleases da família RNase II/RNB: Dis3, Dis3L1 e Dis3L2. Os dois primeiros associam-se ao exossoma, enquanto a Dis3L2 participa numa via alternativa de degradação dependente da 3’-uridilação por uridilil transferases terminais e poli(U) polimerases. Atualmente, a Dis3L2 já foi associada a processos celulares pertinentes como o controlo de qualidade do RNA, e a proliferação e diferenciação celulares. Defeitos na DIS3L2 humana foram relacionados com vários cancros, bem como com a Síndrome de Perlman, uma doença rara de sobre-crescimento fetal que aumenta o risco para o Tumor de Wilms, um tumor renal geralmente encontrado em crianças. Contudo, ainda muito se desconhece do mecanismo de ação da Dis3L2 e da sua preferência distintiva por substratos uridilados. Usando a levedura de fissão como modelo de microrganismo eucariótico para estudar esta ribonuclease extremamente relevante para doenças humanas, este trabalho pretendeu identificar aminoácidos da Dis3L2 de Schizosaccharomyces pombe (SpDis3L2) potencialmente cruciais para a sua atividade, especificidade de substrato, e, em particular, pela preferência por substratos uridilados. Assim, a SpDis3L2 selvagem e respetivos mutantes pontuais foram sobrexpressos e purificados, após a otimização das condições de purificação para que favorecessem a purificação da proteína no seu estado íntegro. A atividade exorribonucleolítica das versões da SpDis3L2 foi analisada sobre diversos substratos de RNA, tendo-se revelado que, à exceção do mutante inativo usado como controlo, todos os mutantes testados eram ativos. Comparando com a proteína SpDis3L2 selvagem, houve mutantes para os quais se observaram diferenças relativamente ao seu nível de atividade e à preferência por substratos uridilados. Tomando estes resultados em consideração, este trabalho constitui o primeiro passo para que se possa, futuramente, analisar versões desta ribonuclease com combinações destas mutações pontuais

TUT7 catalyzes the uridylation of the 3′ end for rapid degradation of histone mRNA

The replication-dependent histone mRNAs end in a stem–loop instead of the poly(A) tail present at the 3′ end of all other cellular mRNAs. Following processing, the 3′ end of histone mRNAs is trimmed to 3 nucleotides (nt) after the stem–loop, and this length is maintained by addition of nontemplated uridines if the mRNA is further trimmed by 3′hExo. These mRNAs are tightly cell-cycle regulated, and a critical regulatory step is rapid degradation of the histone mRNAs when DNA replication is inhibited. An initial step in histone mRNA degradation is digestion 2–4 nt into the stem by 3′hExo and uridylation of this intermediate. The mRNA is then subsequently degraded by the exosome, with stalled intermediates being uridylated. The enzyme(s) responsible for oligouridylation of histone mRNAs have not been definitively identified. Using high-throughput sequencing of histone mRNAs and degradation intermediates, we find that knockdown of TUT7 reduces both the uridylation at the 3′ end as well as uridylation of the major degradation intermediate in the stem. In contrast, knockdown of TUT4 did not alter the uridylation pattern at the 3′ end and had a small effect on uridylation in the stem–loop during histone mRNA degradation. Knockdown of 3′hExo also altered the uridylation of histone mRNAs, suggesting that TUT7 and 3′hExo function together in trimming and uridylating histone mRNAs

A role for the Perlman syndrome exonuclease Dis3l2 in the Lin28-let-7 pathway

The pluripotency factor Lin28 blocks the expression of let-7 microRNAs (miRNAs) in undifferentiated cells during development and functions as an oncogene in a subset of cancers1. Lin28 binds to let-7 precursor RNAs and recruits 3′ terminal uridylyl transferases (TUTases) to selectively inhibit let-7 biogenesis2–4. Uridylated pre-let-7 is refractory to processing by Dicer and is rapidly degraded by an unknown ribonuclease5. Here we identify Dis3l2 as the 3′-5′ exonuclease responsible for the decay of uridylated pre-let-7. Biochemical reconstitution assays reveal that 3′ oligouridylation stimulates Dis3l2 activity in vitro, and knockdown of Dis3l2 in mouse embryonic stem cells leads to the stabilization of pre-let-7. Our study establishes 3′ oligouridylation as an RNA decay signal for Dis3l2 and identifies the first physiological RNA substrate of this novel exonuclease that is mutated in the Perlman syndrome of fetal overgrowth and predisposition to Wilms’ tumor6

Roles for Terminal Uridyl Transferases in the Post-Transcriptional Regulation of Developmental miRNAs

MicroRNAs (miRNAs) are a diverse and evolutionarily conserved class of non-coding RNAs that play a multitude of roles in many branches of eukaryotic biology. The regulation of miRNAs is dynamically controlled both spatially and temporally, and the expression of miRNAs can be modulated at the level of transcription or at points downstream of the miRNA maturation process. A relevant example of post-transcriptional miRNA regulation is the blockade of let-7 precursor miRNAs by Lin28 in embryonic stem cells. This pathway, which is initiated by the small RNA-binding protein Lin28, recruits the terminal uridyl transferase (TUTase) Zcchc11 to add a non-templated oligouridine tail to the miRNAs 3' end, and signals it for degradation by the cytoplasmic exonuclease Dis3l2. The Lin28/let-7 axis is essential for development and metabolic homeostasis, and is reactivated in a subset of human cancers. This thesis describes the biochemical mechanism underlying Lin28-mediated degradation of let-7, as well as a novel role for Zcchc11 and the related TUTase Zcchc6 in targeting mature developmental miRNAs in a Lin28-independent manner

Large-scale analysis of microRNA expression, epi-transcriptomic features and biogenesis.

MicroRNAs are important genetic regulators in both animals and plants. They have a range of functions spanning development, differentiation, growth, metabolism and disease. The advent of next-generation sequencing technologies has made it a relatively straightforward task to detect these molecules and their relative expression via sequencing. There are a large number of published studies with deposited datasets. However, there are currently few resources that capitalize on these data to better understand the features, distribution and biogenesis of miRNAs. Herein, we focus on Human and Mouse for which the majority of data are available. We reanalyse sequencing data from 461 samples into a coordinated catalog of microRNA expression. We use this to perform large-scale analyses of miRNA function and biogenesis. These analyses include global expression comparison, co-expression of miRNA clusters and the prediction of miRNA strand-specificity and underlying constraints. Additionally, we report for the first time a global analysis of miRNA epi-transcriptomic modifications and assess their prevalence across tissues, samples and families. Finally, we report a list of potentially mis-annotated miRNAs in miRBase based on their aggregated modification profiles. The results have been collated into a comprehensive online repository of miRNA expression and features such as modifications and RNA editing events, which is available at: http://wwwdev.ebi.ac.uk/enright-dev/miratlas. We believe these findings will further contribute to our understanding of miRNA function in animals and benefit the miRNA community in general

3′ RNA Uridylation in Epitranscriptomics, Gene Regulation, and Disease

Emerging evidence implicates a wide range of post-transcriptional RNA modifications that play crucial roles in fundamental biological processes including regulating gene expression. Collectively, they are known as epitranscriptomics. Recent studies implicate 3′ RNA uridylation, the non-templated addition of uridine(s) to the terminal end of RNA, as a key player in epitranscriptomics. In this review, we describe the functional roles and significance of 3′ terminal RNA uridylation that has diverse functions in regulating both mRNAs and non-coding RNAs. In mammals, three Terminal Uridylyl Transferases (TUTases) are primarily responsible for 3′ RNA uridylation. These enzymes are also referred to as polyU polymerases. TUTase 1 (TUT1) is implicated in U6 snRNA maturation via uridylation. The TUTases TUT4 and/or TUT7 are the predominant mediators of all other cellular uridylation. Terminal uridylation promotes turnover for many polyadenylated mRNAs, replication-dependent histone mRNAs that lack polyA-tails, and aberrant structured noncoding RNAs. In addition, uridylation regulates biogenesis of a subset of microRNAs and generates isomiRs, sequent variant microRNAs that have altered function in specific cases. For example, the RNA binding protein and proto-oncogene LIN28A and TUT4 work together to polyuridylate pre-let-7, thereby blocking biogenesis and function of the tumor suppressor let-7 microRNA family. In contrast, monouridylation of Group II pre-miRNAs creates an optimal 3′ overhang that promotes recognition and subsequent cleavage by the Dicer-TRBP complex that then yields the mature microRNA. Also, uridylation may play a role in non-canonical microRNA biogenesis. The overall significance of 3′ RNA uridylation is discussed with an emphasis on mammalian development, gene regulation, and disease, including cancer and Perlman syndrome. We also introduce recent changes to the HUGO-approved gene names for multiple terminal nucleotidyl transferases that affects in part TUTase nomenclature (TUT1/TENT1, TENT2/PAPD4/GLD2, TUT4/ZCCHC11/TENT3A, TUT7/ZCCHC6/TENT3B, TENT4A/PAPD7, TENT4B/PAPD5, TENT5A/FAM46A, TENT5B/FAM46B, TENT5C/FAM46C, TENT5D/FAM46D, MTPAP/TENT6/PAPD1)