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2.
Life Sci Alliance ; 5(9)2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35512829

RESUMO

miR-486 is a muscle-enriched microRNA, or "myomiR," that has reduced expression correlated with Duchenne muscular dystrophy (DMD). To determine the function of miR-486 in normal and dystrophin-deficient muscles and elucidate miR-486 target transcripts in skeletal muscle, we characterized mir-486 knockout mice (mir-486 KO). mir-486 KO mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased cardiac fibrosis, and metabolic defects were exacerbated in mir-486 KO:mdx 5cv (DKO) mice. To identify direct in vivo miR-486 muscle target transcripts, we integrated RNA sequencing and chimeric miRNA eCLIP sequencing to identify key transcripts and pathways that contribute towards mir-486 KO and dystrophic disease pathologies. These targets included known and novel muscle metabolic and dystrophic structural remodeling factors of muscle and skeletal muscle contractile transcript targets. Together, our studies identify miR-486 as essential for normal muscle function, a driver of pathological remodeling in dystrophin-deficient muscle, a useful biomarker for dystrophic disease progression, and highlight the use of multiple omic platforms to identify in vivo microRNA target transcripts.


Assuntos
Distrofina , MicroRNAs , Animais , Distrofina/genética , Camundongos , Camundongos Endogâmicos mdx , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Transcriptoma/genética
3.
J Reconstr Microsurg ; 2022 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-35477114

RESUMO

BACKGROUND: Perforators are typically found in rows in the deep inferior epigastric perforator (DIEP) flap. As methods to assess flap perfusion continue to improve, surgeons may be more likely to select perforators traditionally avoided. The purpose of this article is to describe clinical outcomes based on row and number of perforators to reevaluate flap and abdominal donor site morbidity. METHODS: A retrospective analysis was performed on patients who underwent breast reconstruction with DIEP flaps by four microsurgeons from 2013 to 2020. The row and number of perforators were determined from operative reports. Chi-square and t-test or nonparametric Fisher's exact test and Wilcoxon two-sample test were used for discrete and continuous variable, respectively, as applicable. Logistic regression was used for multivariable analyses. RESULTS: Of 628 flaps, 305 were medial row (58.7%), 159 were lateral row (30.6%), and 55 had both rows (10.6%). Partial flap loss was higher in both rows (p = 0.003). Fat necrosis was higher with medial (p = 0.03) and both rows (p = 0.01) when compared with lateral using multivariable analysis. Hernia or bulge was higher in lateral row flaps (lateral: 8/157, 5.1%; medial, 5/299, 1.7%; both, 0/55; p = 0.05); however, mesh was more commonly used in both row flaps (p = 0.05). There was no difference in fat necrosis or abdominal morbidity between single and multiple perforators. CONCLUSION: There was no difference in fat necrosis based on the number or row of perforators. The lateral row provides adequate perfusion but may be associated with an elevated risk of hernia or bulge. Patients may benefit from mesh, especially when both rows are dissected.

4.
JAMA Surg ; 157(5): 436-444, 2022 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-35262660

RESUMO

Importance: Laparoscopic and robotic techniques have both been well adopted as safe options in selected patients undergoing hepatectomy. However, it is unknown whether either approach is superior, especially for major hepatectomy such as right hepatectomy or extended right hepatectomy (RH/ERH). Objective: To compare the outcomes of robotic vs laparoscopic RH/ERH. Design, Setting, and Participants: In this case-control study, propensity score matching analysis was performed to minimize selection bias. Patients undergoing robotic or laparoscopic RH/EHR at 29 international centers from 2008 to 2020 were included. Interventions: Robotic vs laparoscopic RH/ERH. Main Outcomes and Measures: Data on patient demographics, tumor characteristics, and short-term perioperative outcomes were collected and analyzed. Results: Of 989 individuals who met study criteria, 220 underwent robotic and 769 underwent laparoscopic surgery. The median (IQR) age in the robotic RH/ERH group was 61.00 (51.86-69.00) years and in the laparoscopic RH/ERH group was 62.00 (52.03-70.00) years. Propensity score matching resulted in 220 matched pairs for further analysis. Patients' demographics and tumor characteristics were comparable in the matched cohorts. Robotic RH/ERH was associated with a lower open conversion rate (19 of 220 [8.6%] vs 39 of 220 [17.1%]; P = .01) and a shorter postoperative hospital stay (median [IQR], 7.0 [5.0-10.0] days; mean [SD], 9.11 [7.52] days vs median [IQR], 7.0 [5.75-10.0] days; mean [SD], 9.94 [8.99] days; P = .048). On subset analysis of cases performed between 2015 and 2020 after a center's learning curve (50 cases), robotic RH/ERH was associated with a shorter postoperative hospital stay (median [IQR], 6.0 [5.0-9.0] days vs 7.0 [6.0-9.75] days; P = .04) with a similar conversion rate (12 of 220 [7.6%] vs 17 of 220 [10.8%]; P = .46). Conclusion and Relevance: Robotic RH/ERH was associated with a lower open conversion rate and shorter postoperative hospital stay compared with laparoscopic RH/ERH. The difference in open conversion rate was associated with a significant decrease for laparoscopic but not robotic RH/ERH after a center had mounted the learning curve. Use of robotic platform may help to overcome the initial challenges of minimally invasive RH/ERH.

5.
Front Genet ; 13: 848626, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35281806

RESUMO

The process of alternative polyadenylation (APA) generates multiple 3' UTR isoforms for a given locus, which can alter regulatory capacity and on occasion change coding potential. APA was initially characterized for a few genes, but in the past decade, has been found to be the rule for metazoan genes. While numerous differences in APA profiles have been catalogued across genetic conditions, perturbations, and diseases, our knowledge of APA mechanisms and biology is far from complete. In this review, we highlight recent findings regarding the role of the conserved ELAV/Hu family of RNA binding proteins (RBPs) in generating the broad landscape of lengthened 3' UTRs that is characteristic of neurons. We relate this to their established roles in alternative splicing, and summarize ongoing directions that will further elucidate the molecular strategies for neural APA, the in vivo functions of ELAV/Hu RBPs, and the phenotypic consequences of these regulatory paradigms in neurons.

6.
Microsurgery ; 2022 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-35238069

RESUMO

BACKGROUND: Retrospective studies evaluating tissue oximetry in a more recent cohort have shown superiority in flap outcomes. This study compares the use of tissue oximetry in a historical cohort to clinical observation and handheld doppler in a more recent cohort. We hypothesize that there is no benefit to using tissue oximetry. METHODS: A retrospective review was performed on patients who underwent abdominal-based autologous breast reconstruction by five microsurgeons at an academic institution from 2009 to 2020. Method of postoperative flap monitoring was determined then operative details and complications were analyzed. RESULTS: 1367 flaps were reviewed; 740 flaps in 460 patients were monitored with clinical observation and tissue oximetry, and 627 flaps in 391 patients were monitored with clinical observation and handheld doppler. There were no statistical differences in ischemic (p = .59) or congestive complications (p = .41), flap salvage rates when exploring for venous or arterial compromise (p = .52), or early flap loss (p = .56). Although not significant, acute flap-related return to the operating room was lower in the doppler group (4.6%) compared to the oximetry group (6.1%; p = .22). Flaps monitored with tissue oximetry had a statistical increase in length of stay (4.8 ± 1.4 days vs. 3.8 ± 1.6 days; p ≤ .001). The rates of late partial flap loss and fat necrosis were significantly higher in the oximetry group (2.6%, 19/740 vs. 0.3%, 2/740; p = .04) and (18.2%, 135/740 vs. 13.6%, 85/627; p = .02), respectively. CONCLUSIONS: There is no statistical benefit to the use of tissue oximetry compared to handheld doppler in flap monitoring with regards to flap outcomes.

7.
Genes Dev ; 36(3-4): 225-240, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35144965

RESUMO

The BEN domain is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. Here, we characterize several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. We conducted detailed functional, genomic, and structural studies of BEND3. We show that BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. We obtained a high-resolution structure of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with our previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.


Assuntos
Proteínas Repressoras , Fatores de Transcrição , Animais , Sítios de Ligação , Drosophila/metabolismo , Mamíferos , Ligação Proteica , Domínios Proteicos , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo
8.
iScience ; 25(3): 103968, 2022 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-35224469

RESUMO

As the emergence of SARS-CoV-2 variants brings the global pandemic to new levels, the performance of current rapid antigen tests against variants of concern and interest (VOC/I) is of significant public health concern. Here, we report assessment of the Abbot BinaxNOW COVID-19 Antigen Self-Test. Using genetically sequenced remnant clinical samples collected from individuals positive for SARS-CoV-2, we assessed the performance of BinaxNOW against the variants that currently pose public health threats. We measured the limit of detection of BinaxNOW against various VOC/I in a blinded manner. BinaxNOW successfully detected the Omicron (B.1.1.529), Mu (B.1.621), Delta (B.1.617.2), Lambda (C.37), Gamma (P.1), Alpha (B.1.1.7), Beta (B.1.351), Eta (B.1.525), and P.2 variants and at low viral concentrations. BinaxNOW also detected the Omicron variant in individual remnant clinical samples. Overall, these data indicate that this inexpensive and simple-to-use, FDA-authorized and broadly distributed rapid test can reliably detect Omicron, Delta, and other VOC/I.

9.
J Am Med Dir Assoc ; 23(5): 858-864.e5, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-34555338

RESUMO

OBJECTIVES: It is inconsistent in the literature on whether inequalities of health in older age widen or narrow over time. We assessed the associations of socioeconomic status (SES), physical functioning, and mortality in an older age cohort in Hong Kong. DESIGN: Longitudinal cohort study. SETTING AND PARTICIPANTS: We recruited 2032 older adults aged 70+ in 1991 to 1992 and followed them for 10 years. METHODS: SES was operationalized as education, baseline individual income, and longest-held occupation in lifetime. Physical functioning was measured by Barthel's Index for activities of daily living (ADL), from which disability was defined as ADL score <20. Mortality data were obtained from the Death Registry. Bayesian joint modeling with 2 sub-models, mixed-effect, and Cox proportional hazard model, were used to respectively model the associations of SES and disability, and SES and mortality, accounting for selection by mortality. RESULTS: Education and income at baseline were not clearly related to disability, but those with lower education level and income at baseline tended to have their risks increased with time. Older adults who had been mostly economically inactive or unemployed in their lifetime had higher risk of disability [odds ratio 3.24; 95% credible interval (95%CrI) 1.29 to 7.97], and such risk increased over time. For mortality, older adults with no schooling were at higher risk compared with those with secondary education or above (hazard ratio 1.25; 95%CrI 1.00 to 1.57). Income at baseline and longest-held occupation in lifetime were not clearly related to mortality. CONCLUSIONS AND IMPLICATIONS: We observed inequalities of health of older adults in Hong Kong that widened as they age. Community and medical interventions targeting the older adults with the lowest SES would be important to prevent their more rapid decline in physical functioning.

10.
Life Sci Alliance ; 5(1)2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34759052

RESUMO

Intronic ratchet points (RPs) are abundant within long introns in the Drosophila genome and consist of juxtaposed splice acceptor and splice donor (SD) sites. Although they appear to encompass zero-nucleotide exons, we recently clarified that intronic recursive splicing (RS) requires a cryptic exon at the RP (an RS-exon), which is subsequently always skipped and thus absent from mRNA. In addition, Drosophila encodes a smaller set of expressed exons bearing features of RS. Here, we investigate mechanisms that regulate the choice between RP and RS-exon SDs. First, analysis of Drosophila RP SD mutants demonstrates that SD competition suppresses inclusion of cryptic exons in endogenous contexts. Second, characterization of RS-exon reporters implicates exonic sequences as influencing choice of RS-exon usage. Using RS-exon swap and mutagenesis assays, we show exonic sequences can determine RS-exon inclusion. Finally, we provide evidence that splicing can suppress utilization of RP SDs to enable RS-exon expression. Overall, multiple factors can influence splicing of Drosophila RS-exons, which usually result in their complete suppression as zero-nucleotide RPs, but occasionally yield translated RS-exons.


Assuntos
Regulação da Expressão Gênica , Splicing de RNA , Processamento Alternativo , Animais , Sequência de Bases , Drosophila/genética , Éxons , Íntrons , Mutagênese , Sítios de Splice de RNA , Sequências Reguladoras de Ácido Nucleico
11.
Nat Ecol Evol ; 5(12): 1613-1623, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34862477

RESUMO

Meiotic drivers are a class of selfish genetic elements whose existence is frequently hidden due to concomitant suppressor systems. Accordingly, we know little of their evolutionary breadth and molecular mechanisms. Here, we trace the evolution of the Dox meiotic drive system in Drosophila simulans, which affects male-female balance (sex ratio). Dox emerged via stepwise mobilization and acquisition of multiple D. melanogaster gene segments including from protamine, which mediates compaction of sperm chromatin. Moreover, we reveal novel Dox homologs and massive amplification of Dox superfamily genes on X chromosomes of its closest sisters D. mauritiana and D. sechellia. Emergence of Dox loci is tightly associated with 359-class satellite repeats that flank de novo genomic copies. In concert, we find coordinated diversification of autosomal hairpin RNA-class siRNA loci that target subsets of Dox superfamily genes. Overall, we reveal fierce genetic arms races between meiotic drive factors and siRNA suppressors associated with recent speciation.


Assuntos
Drosophila melanogaster , Drosophila , Animais , Drosophila/genética , Drosophila melanogaster/genética , Evolução Molecular , Feminino , Masculino , Meiose , Cromossomo X
12.
Asia Pac Psychiatry ; : e12505, 2021 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-34898030

RESUMO

INTRODUCTION: This study aimed to analyze the estimated prevalence of mental disorders among offenders and compare the estimated crime rate between mentally ill patients and the total population in Hong Kong. METHODS: Service data of offenders referred to psychiatrists at the Siu Lam Psychiatric Centre from January 2011 to December 2020 were analyzed. Demographic data of gender, age on admission, educational level, principal psychiatric diagnosis, index offense, and assessment outcome were collected. RESULTS: Data of 7535 offenders (74.8% males) aged 14 to 97 (mean: 41.3 ± 13.7) years were analyzed. More than 60% (66.2%) had a diagnosable mental disorder. The most prevalent principal psychiatric diagnosis was schizophrenia and related disorder (22.8%), followed by mental and behavioral disorders due to psychoactive substance use (18.6%), and mood disorders (8.8%). The commonest index offenses were theft and related offenses (20.5%), followed by acts intended to cause injury (19.7%), and illicit drug offenses (11.6%). The estimated prevalence of mental illness among prison population was 7.1% (male: 8.2%, female: 5.0%). The estimated crime rate for mentally ill patients was found to be 43.3 to 263.2 per 100 000 population. DISCUSSION: The estimated prevalence of mental disorders among offenders and the estimated crime rate for mentally ill patients are relatively low in Hong Kong. The result was an important effort to document the changing characteristics of mentally ill offenders and provide an estimation of the prevalence and crime rate for mentally ill patients in Hong Kong.

13.
Thorax ; 2021 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-34650003

RESUMO

OBJECTIVE: To determine how early-life risk factors explain socioeconomic inequalities in persistent asthma in adolescence. METHODS: We did a causal mediation analysis using data from 7487 children and young people in the UK Millennium Cohort Study. Persistent asthma was defined as having a diagnosis reported at any two or more time points at 7, 11 or 14 years. The main exposure was maternal education, a measure of early-life socioeconomic circumstances (SECs), used to calculate the relative index of inequality. We assessed how blocks of perinatal (maternal health behaviours, infant characteristics and duration of breastfeeding, measured at 9 months) and environmental risk factors (family housing conditions; potential exposure to infections through childcare type and sibling number, and neighbourhood characteristics, measured at 3 years) mediated the total effect of childhood SECs on persistent asthma risk, calculating the proportion mediated and natural indirect effect (NIE) via blocks of mediators. RESULTS: At age 14 the overall prevalence of persistent asthma was 15%. Children of mothers with lower educational qualifications were more likely to have persistent asthma, with a clear social gradient (degree plus: 12.8% vs no qualifications: 20.3%). The NIE gives the effect of SECs acting only via the mediators and shows a 31% increased odds of persistent asthma when SECs are fixed at the highest level, and mediators at the level which would naturally occur at the lowest SECs versus highest SECs (NIE OR 1.31, 95% CI 1.04 to 1.65). Overall, 58.9% (95% CI 52.9 to 63.7) of the total effect (OR 1.70, 95% CI 1.20 to 2.40) of SECs on risk of persistent asthma in adolescence was mediated by perinatal and environmental characteristics. CONCLUSIONS: Perinatal characteristics and the home environment in early life are more important in explaining socioeconomic inequalities in persistent asthma in British adolescents than more distal environmental exposures outside the home.

14.
Cell Rep ; 36(1): 109335, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34233178

RESUMO

Virgin females of many species conduct distinctive behaviors, compared with post-mated and/or pregnant individuals. In Drosophila, this post-mating switch is initiated by seminal factors, implying that the default female state is virgin. However, we recently showed that loss of miR-iab-4/8-mediated repression of the transcription factor Homothorax (Hth) within the abdominal ventral nerve cord (VNC) causes virgins to execute mated behaviors. Here, we use genomic analysis of mir-iab-4/8 deletion and hth-microRNA (miRNA) binding site mutants (hth[BSmut]) to elucidate doublesex (dsx) as a critical downstream factor. Dsx and Hth proteins are highly complementary in CNS, and Dsx is downregulated in miRNA/hth[BSmut] mutants. Moreover, virgin behavior is highly dose sensitive to developmental dsx function. Strikingly, depletion of Dsx from very restricted abdominal neurons (SAG-1 cells) abrogates female virgin conducts, in favor of mated behaviors. Thus, a double-negative regulatory pathway in the VNC (miR-iab-4/8 ˧ Hth ˧ Dsx) specifies the virgin behavioral state.


Assuntos
Drosophila melanogaster/genética , Redes Reguladoras de Genes , Comportamento Sexual Animal/fisiologia , Abdome/inervação , Animais , Sítios de Ligação , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Larva/genética , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação/genética , Transcriptoma/genética
15.
Minerva Surg ; 76(3): 229-234, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34080820

RESUMO

INTRODUCTION: Near-infrared fluorescent cholangiography (NIFC) using indocyanine green (ICG) is increasingly used to aid in the identification of extrahepatic biliary anatomy. The use of ICG cholangiography for laparoscopic cholecystectomy is suggested to be safe and feasible. This article aimed at reviewing the dosage and timing of the intravenous administration of ICG, its efficacy and potential usage. EVIDENCE ACQUISITION: MEDLINE and PubMed searches were performed using the key words "fluorescent cholangiography," "ICG cholangiography," "near-infrared fluorescent cholangiography" and "laparoscopic cholecystectomy" to identify relevant articles published in English during the years of 2010 to 2020. Reference lists from the articles were reviewed to identify additional pertinent articles. EVIDENCE SYNTHESIS: Several factors can influence the quality of the fluorescence imaging, including the dose and timing of ICG injection, liver function, the thickness of fatty tissue and the presence of inflamed tissues due to acute pathology. Various devices tested also have a different sensitivity to the fluorescence signal. RCTs showed fluorescence cholangiography were comparable to traditional intraoperative cholangiogram in visualizing the extrahepatic biliary anatomy. However, there is still no consensus in the dosing of ICG and the time interval between ICG injection and detection of biliary fluorescence. Fluorescence cholangiography's ability to enhance such visualization can potentially reduce bile duct injury risks and shorten the operative time. However, no valuable data for bile duct injury prevention or detection could be retrieved. CONCLUSIONS: NIFC is demonstrated as a safe, non-irradiating technique to identify and aid in the visualization of extrahepatic biliary anatomy. Laparoscopic cholecystectomy with real-time NIFC enables a better visualization and identification of biliary anatomy and therefore it is potentially as a means of increasing the safety of laparoscopic cholecystectomy. Whether this translates into reducing complication rates must still be determined. The dosage and timing of the intravenous administration of ICG relative to the operative procedure still requires optimization to ensure reliable images.


Assuntos
Sistema Biliar , Colecistectomia Laparoscópica , Colangiografia , Corantes , Humanos , Verde de Indocianina
16.
PLoS Genet ; 17(5): e1009563, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34033644

RESUMO

Accurate splice site selection is critical for fruitful gene expression. Recently, the mammalian EJC was shown to repress competing, cryptic, splice sites (SS). However, the evolutionary generality of this remains unclear. Here, we demonstrate the Drosophila EJC suppresses hundreds of functional cryptic SS, even though most bear weak splicing motifs and are seemingly incompetent. Mechanistically, the EJC directly conceals cryptic splicing elements by virtue of its position-specific recruitment, preventing aberrant SS definition. Unexpectedly, we discover the EJC inhibits scores of regenerated 5' and 3' recursive SS on segments that have already undergone splicing, and that loss of EJC regulation triggers faulty resplicing of mRNA. An important corollary is that certain intronless cDNA constructs yield unanticipated, truncated transcripts generated by resplicing. We conclude the EJC has conserved roles to defend transcriptome fidelity by (1) repressing illegitimate splice sites on pre-mRNAs, and (2) preventing inadvertent activation of such sites on spliced segments.


Assuntos
Drosophila melanogaster , Éxons , Íntrons , Complexos Multiproteicos , Splicing de RNA , RNA Mensageiro/metabolismo , Processamento Alternativo , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Éxons/genética , Íntrons/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , Transcriptoma
17.
Data Brief ; 36: 107037, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34007867

RESUMO

microRNAs (miRNAs) are a broad class of ~22 nucleotide regulatory RNA, which collectively have broad effects on the transcriptome and are involved in diverse biology, from development and adult physiology, and from homeostasis to disease and pathology. We investigated the effects of systematically expressing microRNAs (miRNAs) during the development of the Drosophila compound eye using the GMR-Gal4 driver. The objective was to determine what fraction of miRNAs were capable of inducing aberrant morphology that was easily and reproducibly scored by visual inspection under a dissecting microscope. We assayed multiple independent insertions of 166 miRNA transgenes (536 lines), comprising solo miRNAs, miRNA operons and individual constituent miRNAs from operons. We find a substantial number reproducibly altered normal eye development and a smaller number induced lethality in most or all progeny. We provide the comprehensive results of this screen, documenting numerous miRNA transgenes that interfered with normal eye development when activated using GMR-Gal4. These data can be mined by the Drosophila community to query the in vivo effects of any individual miRNA of interest in the eye, as well as utilized as a foundation for more complex genetic perturbations that involve miRNA misexpression in the eye.

18.
PLoS Genet ; 17(4): e1009439, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33826609

RESUMO

ELAV/Hu factors are conserved RNA binding proteins (RBPs) that play diverse roles in mRNA processing and regulation. The founding member, Drosophila Elav, was recognized as a vital neural factor 35 years ago. Nevertheless, little was known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3' UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets of Drosophila are known, ectopic expression of each of the three family members (Elav, Fne and Rbp9) alters hundreds of cassette exon and alternative last exon (ALE) splicing choices. Reciprocally, double mutants of elav/fne, but not elav alone, exhibit opposite effects on both classes of regulated mRNA processing events in larval CNS. While manipulation of Drosophila ELAV/Hu RBPs induces both exon skipping and inclusion, characteristic ELAV/Hu motifs are enriched only within introns flanking exons that are suppressed by ELAV/Hu factors. Moreover, the roles of ELAV/Hu factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3' UTR extensions in neurons, since both processes involve bypass of proximal polyadenylation signals linked to ELAV/Hu motifs downstream of cleavage sites. We corroborate the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification in Drosophila and mammalian neurons.


Assuntos
Processamento Alternativo/genética , Diferenciação Celular/genética , Proteínas de Drosophila/genética , Proteínas ELAV/genética , Proteína Semelhante a ELAV 1/genética , Neurônios/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Humanos , Larva/genética , Larva/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Poliadenilação/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transcriptoma/genética
20.
Nat Commun ; 12(1): 1458, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674589

RESUMO

Epitranscriptomic modifications can impact behavior. Here, we used Drosophila melanogaster to study N6-methyladenosine (m6A), the most abundant modification of mRNA. Proteomic and functional analyses confirm its nuclear (Ythdc1) and cytoplasmic (Ythdf) YTH domain proteins as major m6A binders. Assays of short term memory in m6A mutants reveal neural-autonomous requirements of m6A writers working via Ythdf, but not Ythdc1. Furthermore, m6A/Ythdf operate specifically via the mushroom body, the center for associative learning. We map m6A from wild-type and Mettl3 mutant heads, allowing robust discrimination of Mettl3-dependent m6A sites that are highly enriched in 5' UTRs. Genomic analyses indicate that Drosophila m6A is preferentially deposited on genes with low translational efficiency and that m6A does not affect RNA stability. Nevertheless, functional tests indicate a role for m6A/Ythdf in translational activation. Altogether, our molecular genetic analyses and tissue-specific m6A maps reveal selective behavioral and regulatory defects for the Drosophila Mettl3/Ythdf pathway.


Assuntos
Adenosina/análogos & derivados , Adenosina/metabolismo , Drosophila melanogaster/fisiologia , Aprendizagem/fisiologia , Memória/fisiologia , Regiões 5' não Traduzidas , Adenosina/genética , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Feminino , Proteínas Nucleares/metabolismo , Proteômica , Estabilidade de RNA , RNA Mensageiro/metabolismo
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