Motif conservation, stability, and host gene expression are the main drivers of snoRNA expression across vertebrates.

Small nucleolar RNAs (snoRNAs) are structured noncoding RNAs present in multiple copies within eukaryotic genomes. snoRNAs guide chemical modifications on their target RNA and regulate processes like ribosome assembly and splicing. Most human snoRNAs are embedded within host gene introns, the remainder being independently expressed from intergenic regions. We recently characterized the abundance of snoRNAs and their host gene across several healthy human tissues and found that the level of most snoRNAs does not correlate with that of their host gene, with the observation that snoRNAs embedded within the same host gene often differ drastically in abundance. To better understand the determinants of snoRNA expression, we trained machine learning models to predict whether snoRNAs are expressed or not in human tissues based on more than 30 collected features related to snoRNAs and their genomic context. By interpreting the models' predictions, we find that snoRNAs rely on conserved motifs, a stable global structure and terminal stem, and a transcribed locus to be expressed. We observe that these features explain well the varying abundance of snoRNAs embedded within the same host gene. By predicting the expression status of snoRNAs across several vertebrates, we notice that only one-third of all annotated snoRNAs are expressed per genome, as in humans. Our results suggest that ancestral snoRNAs disseminated within vertebrate genomes, sometimes leading to the development of new functions and a probable gain in fitness and thereby conserving features favorable to the expression of these few snoRNAs, the large remainder often degenerating into pseudogenes.

Replisome Proximal Protein Associations and Dynamic Proteomic Changes at Stalled Replication Forks.

DNA replication is a fundamental cellular process that ensures the transfer of genetic information during cell division. Genome duplication takes place in S phase and requires a dynamic and highly coordinated recruitment of multiple proteins at replication forks. Various genotoxic stressors lead to fork instability and collapse, hence the need for DNA repair pathways. By identifying the multitude of protein interactions implicated in those events, we can better grasp the complex and dynamic molecular mechanisms that facilitate DNA replication and repair. Proximity-dependent biotin identification was used to identify associations with 17 proteins within four core replication components, namely the CDC45/MCM2-7/GINS helicase that unwinds DNA, the DNA polymerases, replication protein A subunits, and histone chaperones needed to disassemble and reassemble chromatin. We further investigated the impact of genotoxic stress on these interactions. This analysis revealed a vast proximity association network with 108 nuclear proteins further modulated in the presence of hydroxyurea; 45 being enriched and 63 depleted. Interestingly, hydroxyurea treatment also caused a redistribution of associations with 11 interactors, meaning that the replisome is dynamically reorganized when stressed. The analysis identified several poorly characterized proteins, thereby uncovering new putative players in the cellular response to DNA replication arrest. It also provides a new comprehensive proteomic framework to understand how cells respond to obstacles during DNA replication.

OpenProt 2.0 builds a path to the functional characterization of alternative proteins.

The OpenProt proteogenomic resource (https://www.openprot.org/) provides users with a complete and freely accessible set of non-canonical or alternative open reading frames (AltORFs) within the transcriptome of various species, as well as functional annotations of the corresponding protein sequences not found in standard databases. Enhancements in this update are largely the result of user feedback and include the prediction of structure, subcellular localization, and intrinsic disorder, using cutting-edge algorithms based on machine learning techniques. The mass spectrometry pipeline now integrates a machine learning-based peptide rescoring method to improve peptide identification. We continue to help users explore this cryptic proteome by providing OpenCustomDB, a tool that enables users to build their own customized protein databases, and OpenVar, a genomic annotator including genetic variants within AltORFs and protein sequences. A new interface improves the visualization of all functional annotations, including a spectral viewer and the prediction of multicoding genes. All data on OpenProt are freely available and downloadable. Overall, OpenProt continues to establish itself as an important resource for the exploration and study of new proteins.

snoDB 2.0: an enhanced interactive database, specializing in human snoRNAs.

snoDB is an interactive database of human small nucleolar RNAs (snoRNAs) that includes up-to-date information on snoRNA features, genomic location, conservation, host gene, snoRNA-RNA targets and snoRNA abundance and provides links to other resources. In the second edition of this database (snoDB 2.0), we added an entirely new section on ribosomal RNA (rRNA) chemical modifications guided by snoRNAs with easy navigation between the different rRNA versions used in the literature and experimentally measured levels of modification. We also included new layers of information, including snoRNA motifs, secondary structure prediction, snoRNA-protein interactions, copy annotations and low structure bias expression data in a wide panel of tissues and cell lines to bolster functional probing of snoRNA biology. Version 2.0 features updated identifiers, more links to external resources and duplicate entry resolution. As a result, snoDB 2.0, which is freely available at https://bioinfo-scottgroup.med.usherbrooke.ca/snoDB/, represents a one-stop shop for snoRNA features, rRNA modification targets, functional impact and potential regulators.

The regulatory roles of small nucleolar RNAs within their host locus.

Small nucleolar RNAs (snoRNAs) are a class of conserved noncoding RNAs forming complexes with proteins to catalyse site-specific modifications on ribosomal RNA. Besides this canonical role, several snoRNAs are now known to regulate diverse levels of gene expression. While these functions are carried out in trans by mature snoRNAs, evidence has also been emerging of regulatory roles of snoRNAs in cis, either within their genomic locus or as longer transcription intermediates during their maturation. Herein, we review recent findings that snoRNAs can interact in cis with their intron to regulate the expression of their host gene. We also explore the ever-growing diversity of longer host-derived snoRNA extensions and their functional impact across the transcriptome. Finally, we discuss the role of snoRNA duplications into forging these new layers of snoRNA-mediated regulation, as well as their involvement in the genomic imprinting of their host locus.

The snoGloBe interaction predictor reveals a broad spectrum of C/D snoRNA RNA targets.

Box C/D small nucleolar RNAs (snoRNAs) are a conserved class of RNA known for their role in guiding ribosomal RNA 2'-O-ribose methylation. Recently, C/D snoRNAs were also implicated in regulating the expression of non-ribosomal genes through different modes of binding. Large scale RNA-RNA interaction datasets detect many snoRNAs binding messenger RNA, but are limited by specific experimental conditions. To enable a more comprehensive study of C/D snoRNA interactions, we created snoGloBe, a human C/D snoRNA interaction predictor based on a gradient boosting classifier. SnoGloBe considers the target type, position and sequence of the interactions, enabling it to outperform existing predictors. Interestingly, for specific snoRNAs, snoGloBe identifies strong enrichment of interactions near gene expression regulatory elements including splice sites. Abundance and splicing of predicted targets were altered upon the knockdown of their associated snoRNA. Strikingly, the predicted snoRNA interactions often overlap with the binding sites of functionally related RNA binding proteins, reinforcing their role in gene expression regulation. SnoGloBe is also an excellent tool for discovering viral RNA targets, as shown by its capacity to identify snoRNAs targeting the heavily methylated SARS-CoV-2 RNA. Overall, snoGloBe is capable of identifying experimentally validated binding sites and predicting novel sites with shared regulatory function.

Current RNA-seq methodology reporting limits reproducibility.

Ribonucleic acid sequencing (RNA-seq) identifies and quantifies RNA molecules from a biological sample. Transformation from raw sequencing data to meaningful gene or isoform counts requires an in silico bioinformatics pipeline. Such pipelines are modular in nature, built using selected software and biological references. Software is usually chosen and parameterized according to the sequencing protocol and biological question. However, while biological and technical noise is alleviated through replicates, biases due to the pipeline and choice of biological references are often overlooked. Here, we show that the current standard practice prevents reproducibility in RNA-seq studies by failing to specify required methodological information. Peer-reviewed articles are intended to apply currently accepted scientific and methodological standards. Inasmuch as the bias-less and optimal RNA-seq pipeline is not perfectly defined, methodological information holds a meaningful role in defining the results. This work illustrates the need for a standardized and explicit display of methodological information in RNA-seq experiments.

RBFOX2 alters splicing outcome in distinct binding modes with multiple protein partners.

RBFOX2 controls the splicing of a large number of transcripts implicated in cell differentiation and development. Parsing RNA-binding protein datasets, we uncover that RBFOX2 can interact with hnRNPC, hnRNPM and SRSF1 to regulate splicing of a broad range of splicing events using different sequence motifs and binding modes. Using immunoprecipitation, specific RBP knockdown, RNA-seq and splice-sensitive PCR, we show that RBFOX2 can target splice sites using three binding configurations: single, multiple or secondary modes. In the single binding mode RBFOX2 is recruited to its target splice sites through a single canonical binding motif, while in the multiple binding mode RBFOX2 binding sites include the adjacent binding of at least one other RNA binding protein partner. Finally, in the secondary binding mode RBFOX2 likely does not bind the RNA directly but is recruited to splice sites lacking its canonical binding motif through the binding of one of its protein partners. These dynamic modes bind distinct sets of transcripts at different positions and distances relative to alternative splice sites explaining the heterogeneity of RBFOX2 targets and splicing outcomes.

OpenProt 2021: deeper functional annotation of the coding potential of eukaryotic genomes.

OpenProt (www.openprot.org) is the first proteogenomic resource supporting a polycistronic annotation model for eukaryotic genomes. It provides a deeper annotation of open reading frames (ORFs) while mining experimental data for supporting evidence using cutting-edge algorithms. This update presents the major improvements since the initial release of OpenProt. All species support recent NCBI RefSeq and Ensembl annotations, with changes in annotations being reported in OpenProt. Using the 131 ribosome profiling datasets re-analysed by OpenProt to date, non-AUG initiation starts are reported alongside a confidence score of the initiating codon. From the 177 mass spectrometry datasets re-analysed by OpenProt to date, the unicity of the detected peptides is controlled at each implementation. Furthermore, to guide the users, detectability statistics and protein relationships (isoforms) are now reported for each protein. Finally, to foster access to deeper ORF annotation independently of one's bioinformatics skills or computational resources, OpenProt now offers a data analysis platform. Users can submit their dataset for analysis and receive the results from the analysis by OpenProt. All data on OpenProt are freely available and downloadable for each species, the release-based format ensuring a continuous access to the data. Thus, OpenProt enables a more comprehensive annotation of eukaryotic genomes and fosters functional proteomic discoveries.

A Retrospective and Prospective Cohort Study Comparing Pediatric Patients With Cleft Lip and Palate From the United States and Guatemala.

Orofacial clefts (OFC) remain among the most prevalent congenital abnormalities worldwide. In the United States in 2010 to 2014, 16.2 of 10,000 live births are born with OFC compared with 23.6 of 10,000 in Alta Verapaz, Guatemala in 2012. Demographics and cleft severity scores were retrospectively gathered from 514 patients with isolated OFC at the Children's Hospital of Philadelphia scheduled for surgery from 2012 to 2019 and from 115 patients seen during surgical mission trips to Guatemala City from 2017 to 2020. Risk factors were also gathered prospectively from Guatemalan families. The Guatemalan cohort had a significantly lower prevalence of cleft palate only compared with the US cohort, which may be a result of greater cleft severity in the population or poor screening and subsequent increased mortality of untreated cleft palate. Of those with lip involvement, Guatemalan patients were significantly more likely to have complete cleft lip, associated cleft palate, and right-sided and bilateral clefts, demonstrating an increased severity of Guatemalan cleft phenotype. Primary palate and lip repair for the Guatemalan cohort occurred at a significantly older age than that of the US cohort, placing Guatemalan patients at increased risk for long-term complications such as communication difficulties. Potential OFC risk factors identified in the Guatemalan cohort included maternal cooking-fire and agricultural chemical exposure, poor prenatal vitamin intake, poverty, and risk factors related to primarily corn-based diets. OFC patients who primarily rely on surgical missions for cleft care would likely benefit from more comprehensive screening and investigation into risk factors for more severe OFC phenotypes.

Comprehensive Long-Term Outcomes Following Mandibular Distraction Osteogenesis.

OBJECTIVE: To describe long-term outcomes and complications following mandibular distraction osteogenesis (MDO) in a diverse patient cohort. DESIGN: Cross-sectional study. SETTING: Single tertiary-care pediatric center. PATIENTS: Forty-eight patients previously undergoing MDO with minimum 4-year follow-up. MAIN OUTCOME MEASURES: Respiratory outcomes, feeding patterns, dental development, motor/sensory nerve function, temporo-mandibular joint function, and postsurgical scarring. RESULTS: Forty-six patients with a median age of 7 years were evaluated. Of 20 nonsyndromic patients, none required additional airway procedures, none required continuous positive airway pressure (CPAP) during sleep, and 19 (95%) fed exclusively by mouth. Among 26 syndromic patients, 7 (27%) required CPAP and 8 (31%) were tube fed. Permanent first molar differences were seen in the majority of subjects; patterns of damage interfering with function were more common in syndromic (13/28, 46%) compared to nonsyndromic (5/24, 21%; P = .014) subjects. MDO prior to age two was associated with more frequent and worse dental damage (P = .001). Inferior alveolar nerve and marginal mandibular nerve function were fully intact in 37 (80%) and 39 (85%) of patients, respectively. Three patients (6%), all with associated genetic syndromes, demonstrated severe nerve impairment. By the Vancouver scar scale, ≥ 80% of surgical scars were rated in the most favorable category for each quality assessed. Temporomandibular joint dysfunction was rare. CONCLUSIONS: MDO shows highly favorable long-term respiratory, feeding, nerve, and scar outcomes in nonsyndromic patients, although permanent molar changes not precluding tooth viability are commonly seen. Patients with associated syndromes demonstrate respiratory and feeding benefits, but higher rates of dental and nerve abnormalities.

Impact of Illustrated Postoperative Instructions on Knowledge and Retention During a Cleft Lip and Palate Surgical Mission.

OBJECTIVE: To determine the impact of illustrated postoperative instructions on patient-caregiver knowledge and retention. DESIGN: Prospective study with all participants receiving an educational intervention. SETTING: Pediatric plastic surgical missions in Guatemala City, Guatemala, between 2019 and 2020. PARTICIPANTS: A total of 63 majority-indigenous Guatemalan caregivers of patients receiving cleft lip and/or palate surgery. INTERVENTION: Illustrated culturally appropriate postoperative care instructions were iteratively developed and given to caregivers who were surveyed on illustration-based and text-based information at preoperative, postoperative, and four-week follow-up time points. MAIN OUTCOME MEASURE: Postoperative care knowledge of illustration-based versus text-based information as determined by the ability to answer 11 illustration- and 8 text-based all-or-nothing questions, as well as retention of knowledge as determined by the same survey given at four weeks follow-up. RESULTS: Scores for illustration-based and text-based information both significantly increased after caregivers received the postoperative instructions (+13.30 ± 3.78 % SE, + 11.26 ± 4.81 % SE; P < .05). At follow-up, scores were unchanged for illustration-based (-3.42 ± 4.49 % SE, P > .05), but significantly lower for text-based information (-28.46 ± 6.09 % SE, P < .01). Retention of text-based information at follow-up correlated positively with education level and Spanish literacy, but not for illustration-based. CONCLUSIONS: In the setting of language and cultural barriers on a surgical mission, understanding of illustration-based and text-based information both increased after verbal explanation of illustrated postoperative instructions. Illustration-based information was more likely to be retained by patient caregivers after four weeks than text-based information, the latter of which correlated with increased education and literacy.

SAPFIR: A webserver for the identification of alternative protein features.

BACKGROUND: Alternative splicing can increase the diversity of gene functions by generating multiple isoforms with different sequences and functions. However, the extent to which splicing events have functional consequences remains unclear and predicting the impact of splicing events on protein activity is limited to gene-specific analysis. RESULTS: To accelerate the identification of functionally relevant alternative splicing events we created SAPFIR, a predictor of protein features associated with alternative splicing events. This webserver tool uses InterProScan to predict protein features such as functional domains, motifs and sites in the human and mouse genomes and link them to alternative splicing events. Alternative protein features are displayed as functions of the transcripts and splice sites. SAPFIR could be used to analyze proteins generated from a single gene or a group of genes and can directly identify alternative protein features in large sequence data sets. The accuracy and utility of SAPFIR was validated by its ability to rediscover previously validated alternative protein domains. In addition, our de novo analysis of public datasets using SAPFIR indicated that only a small portion of alternative protein domains was conserved between human and mouse, and that in human, genes involved in nervous system process, regulation of DNA-templated transcription and aging are more likely to produce isoforms missing functional domains due to alternative splicing. CONCLUSION: Overall SAPFIR represents a new tool for the rapid identification of functional alternative splicing events and enables the identification of cellular functions affected by a defined splicing program. SAPFIR is freely available at https://bioinfo-scottgroup.med.usherbrooke.ca/sapfir/ , a website implemented in Python, with all major browsers supported. The source code is available at https://github.com/DelongZHOU/SAPFIR .

Human plasma pregnancy-associated miRNAs and their temporal variation within the first trimester of pregnancy.

BACKGROUND: During pregnancy, maternal metabolism undergoes substantial changes to support the developing fetus. Such changes are finely regulated by different mechanisms carried out by effectors such as microRNAs (miRNAs). These small non-coding RNAs regulate numerous biological functions, mostly through post-transcriptional repression of gene expression. miRNAs are also secreted in circulation by numerous organs, such as the placenta. However, the complete plasmatic microtranscriptome of pregnant women has still not been fully described, although some miRNA clusters from the chromosome 14 (C14MC) and the chromosome 19 (C19MC and miR-371-3 cluster) have been proposed as being specific to pregnancy. Our aims were thus to describe the plasma microtranscriptome during the first trimester of pregnancy, by assessing the differences with non-pregnant women, and how it varies between the 4th and the 16th week of pregnancy. METHODS: Plasmatic miRNAs from 436 pregnant (gestational week 4 to 16) and 15 non-pregnant women were quantified using Illumina HiSeq next-generation sequencing platform. Differentially abundant miRNAs were identified using DESeq2 package (FDR q-value ≤ 0.05) and their targeted biological pathways were assessed with DIANA-miRpath. RESULTS: A total of 2101 miRNAs were detected, of which 191 were differentially abundant (fold change < 0.05 or > 2, FDR q-value ≤ 0.05) between pregnant and non-pregnant women. Of these, 100 miRNAs were less and 91 miRNAs were more abundant in pregnant women. Additionally, the abundance of 57 miRNAs varied according to gestational age at first trimester, of which 47 were positively and 10 were negatively associated with advancing gestational age. miRNAs from the C19MC were positively associated with both pregnancy and gestational age variation during the first trimester. Biological pathway analysis revealed that these 191 (pregnancy-specific) and 57 (gestational age markers) miRNAs targeted genes involved in fatty acid metabolism, ECM-receptor interaction and TGF-beta signaling pathways. CONCLUSION: We have identified circulating miRNAs specific to pregnancy and/or that varied with gestational age in first trimester. These miRNAs target biological pathways involved in lipid metabolism as well as placenta and embryo development, suggesting a contribution to the maternal metabolic adaptation to pregnancy and fetal growth.

Downregulation of KRAB zinc finger proteins in 5-fluorouracil resistant colorectal cancer cells.

Radio-chemotherapy with 5-flu orouracil (5-FU) is the standard of care treatment for patients with colorectal cancer, but it is only effective for a third of them. Despite our understanding of the mechanism of action of 5-FU, drug resistance remains a significant limitation to the clinical use of 5-FU, as both intrinsic and acquired chemoresistance represents the major obstacles for the success of 5-FU-based chemotherapy. In order to identify the mechanism of acquired resistance, 5-FU chemoresistance was induced in CRC cell lines by passaging cells with increasing concentrations of 5-FU. To study global molecular changes, quantitative proteomics and transcriptomics analyses were performed on these cell lines, comparing the resistant cells as well as the effect of chemo and radiotherapy. Interestingly, a very high proportion of downregulated genes were annotated as transcription factors coding for Krüppel-associated box (KRAB) domain-containing zinc-finger proteins (KZFPs), the largest family of transcriptional repressors. Among nearly 350 KRAB-ZFPs, almost a quarter were downregulated after the induction of a 5-FU-resistance including a common one between the three CRC cell lines, ZNF649, whose role is still unknown. To confirm the observations of the proteomic and transcriptomic approaches, the abundance of 20 different KZFPs and control mRNAs was validated by RT-qPCR. In fact, several KZFPs were no longer detectable using qPCR in cell lines resistant to 5-FU, and the KZFPs that were downregulated only in one or two cell lines showed similar pattern of expression as measured by the omics approaches. This proteomic, transcriptomic and genomic analysis of intrinsic and acquired resistance highlights a possible new mechanism involved in the cellular adaptation to 5-FU and therefore identifies potential new therapeutic targets to overcome this resistance.

Cardiomyocyte-specific Srsf3 deletion reveals a mitochondrial regulatory role.

Serine-rich splicing factor 3 (SRSF3) was recently reported as being necessary to preserve RNA stability via an mTOR mechanism in a cardiac mouse model in adulthood. Here, we demonstrate the link between Srsf3 and mitochondrial integrity in an embryonic cardiomyocyte-specific Srsf3 conditional knockout (cKO) mouse model. Fifteen-day-old Srsf3 cKO mice showed dramatically reduced (below 50%) survival and reduced the left ventricular systolic performance, and histological analysis of these hearts revealed a significant increase in cardiomyocyte size, confirming the severe remodeling induced by Srsf3 deletion. RNA-seq analysis of the hearts of 5-day-old Srsf3 cKO mice revealed early changes in expression levels and alternative splicing of several transcripts related to mitochondrial integrity and oxidative phosphorylation. Likewise, the levels of several protein complexes of the electron transport chain decreased, and mitochondrial complex I-driven respiration of permeabilized cardiac muscle fibers from the left ventricle was impaired. Furthermore, transmission electron microscopy analysis showed disordered mitochondrial length and cristae structure. Together with its indispensable role in the physiological maintenance of mouse hearts, these results highlight the previously unrecognized function of Srsf3 in regulating the mitochondrial integrity.

Human Hepatocyte Nuclear Factor 4-α Encodes Isoforms with Distinct Transcriptional Functions.

HNF4α is a nuclear receptor produced as 12 isoforms from two promoters by alternative splicing. To characterize the transcriptional capacities of all 12 HNF4α isoforms, stable lines expressing each isoform were generated. The entire transcriptome associated with each isoform was analyzed as well as their respective interacting proteome. Major differences were noted in the transcriptional function of these isoforms. The α1 and α2 isoforms were the strongest regulators of gene expression whereas the α3 isoform exhibited significantly reduced activity. The α4, α5, and α6 isoforms, which use an alternative first exon, were characterized for the first time, and showed a greatly reduced transcriptional potential with an inability to recognize the consensus response element of HNF4α. Several transcription factors and coregulators were identified as potential specific partners for certain HNF4α isoforms. An analysis integrating the vast amount of omics data enabled the identification of transcriptional regulatory mechanisms specific to certain HNF4α isoforms, hence demonstrating the importance of considering all isoforms given their seemingly diverse functions.

Reducing the structure bias of RNA-Seq reveals a large number of non-annotated non-coding RNA.

The study of RNA expression is the fastest growing area of genomic research. However, despite the dramatic increase in the number of sequenced transcriptomes, we still do not have accurate estimates of the number and expression levels of non-coding RNA genes. Non-coding transcripts are often overlooked due to incomplete genome annotation. In this study, we use annotation-independent detection of RNA reads generated using a reverse transcriptase with low structure bias to identify non-coding RNA. Transcripts between 20 and 500 nucleotides were filtered and crosschecked with non-coding RNA annotations revealing 111 non-annotated non-coding RNAs expressed in different cell lines and tissues. Inspecting the sequence and structural features of these transcripts indicated that 60% of these transcripts correspond to new snoRNA and tRNA-like genes. The identified genes exhibited features of their respective families in terms of structure, expression, conservation and response to depletion of interacting proteins. Together, our data reveal a new group of RNA that are difficult to detect using standard gene prediction and RNA sequencing techniques, suggesting that reliance on actual gene annotation and sequencing techniques distorts the perceived architecture of the human transcriptome.

snoDB: an interactive database of human snoRNA sequences, abundance and interactions.

Small nucleolar RNAs (snoRNAs) are an abundant type of non-coding RNA with conserved functions in all known eukaryotes. Classified into two main families, the box C/D and H/ACA snoRNAs, they enact their most well characterized role of guiding site specific modifications in ribosomal RNA, through the formation of specific ribonucleoprotein complexes, with fundamental implications in ribosome biogenesis. However, it is becoming increasingly clear that the landscape of snoRNA cellular functionality is much broader than it once seemed with novel members, non-uniform expression patterns, new and diverse targets as well as several emerging non-canonical functions ranging from the modulation of alternative splicing to the regulation of chromatin architecture. In order to facilitate the further characterization of human snoRNAs in a holistic manner, we introduce an online interactive database tool: snoDB. Its purpose is to consolidate information on human snoRNAs from different sources such as sequence databases, target information, both canonical and non-canonical from the literature and from high-throughput RNA-RNA interaction datasets, as well as high-throughput sequencing data that can be visualized interactively.