High resolution temporal transcriptomics of mouse embryoid body development reveals complex expression dynamics of coding and noncoding loci.

Cellular responses to stimuli are rapid and continuous and yet the vast majority of investigations of transcriptional responses during developmental transitions typically use long interval time courses; limiting the available interpretive power. Moreover, such experiments typically focus on protein-coding transcripts, ignoring the important impact of long noncoding RNAs. We therefore evaluated coding and noncoding expression dynamics at unprecedented temporal resolution (6-hourly) in differentiating mouse embryonic stem cells and report new insight into molecular processes and genome organization. We present a highly resolved differentiation cascade that exhibits coding and noncoding transcriptional alterations, transcription factor network interactions and alternative splicing events, little of which can be resolved by long-interval developmental time-courses. We describe novel short lived and cycling patterns of gene expression and dissect temporally ordered gene expression changes in response to transcription factors. We elucidate patterns in gene co-expression across the genome, describe asynchronous transcription at bidirectional promoters and functionally annotate known and novel regulatory lncRNAs. These findings highlight the complex and dynamic molecular events underlying mammalian differentiation that can only be observed though a temporally resolved time course

Non-coding RNAs modulate function of extracellular matrix proteins

The extracellular matrix (ECM) creates a multifaceted system for the interaction of diverse structural proteins, matricellular molecules, proteoglycans, hyaluronan, and various glycoproteins that collaborate and bind with each other to produce a bioactive polymer. Alterations in the composition and configuration of ECM elements influence the cellular phenotype, thus participating in the pathogenesis of several human disorders. Recent studies indicate the crucial roles of non-coding RNAs in the modulation of ECM. Several miRNAs such as miR-21, miR-26, miR-19, miR-140, miR-29, miR-30, miR-133 have been dysregulated in disorders that are associated with disruption or breakdown of the ECM. Moreover, expression of MALAT1, PVT1, SRA1, n379519, RMRP, PFL, TUG1, TM1P3, FAS-AS1, PART1, XIST, and expression of other lncRNAs is altered in disorders associated with the modification of ECM components. In the current review, we discuss the role of lncRNAs and miRNAs in the modification of ECM and their relevance with the pathophysiology of human disorders such as cardiac/ lung fibrosis, cardiomyopathy, heart failure, asthma, osteoarthritis, and cancers. © 2021 The Author(s

A Pathway to Precision Medicine for Aboriginal Australians: A Study Protocol

Published: 21 June 2021(1) Background: Genomic precision medicine (PM) utilises people’s genomic data to inform the delivery of preventive and therapeutic health care. PM has not been well-established for use with people of Aboriginal and Torres Strait Islander ancestry due to the paucity of genomic data from these communities. We report the development of a new protocol using co-design methods to enhance the potential use of PM for Aboriginal Australians. (2) Methods: This iterative qualitative study consists of five main phases. Phase-I will ensure appropriate governance of the project and establishment of a Project Advisory Committee. Following an initial consultation with the Aboriginal community, Phase-II will invite community members to participate in co-design workshops. In Phase-III, the Chief Investigators will participate in co-design workshops and document generated ideas. The notes shall be analysed thematically in Phase-IV with Aboriginal community representatives, and the summary will be disseminated to the communities. In Phase-V, we will evaluate the co-design process and adapt our protocol for the use in partnership with other communities. (3) Discussion: This study protocol represents a crucial first step to ensure that PM research is relevant and acceptable to Aboriginal Australians. Without fair access to PM, the gap in health outcome between Aboriginal and non-Aboriginal Australians will continue to widen.Yeu-Yao Cheng, Jack Nunn, John Skinner, Boe Rambaldini, Tiffany Boughtwood, Tom Calma, Alex Brown, Cliff Meldrum, Marcel E. Dinger, Jennifer A. Byrne, Debbie McCowen, Jayden Potter, Kerry Faires, Sandra Cooper and Kylie Gwynn

Introme accurately predicts the impact of coding and noncoding variants on gene splicing, with clinical applications

Predicting the impact of coding and noncoding variants on splicing is challenging, particularly in non-canonical splice sites, leading to missed diagnoses in patients. Existing splice prediction tools are complementary but knowing which to use for each splicing context remains difficult. Here, we describe Introme, which uses machine learning to integrate predictions from several splice detection tools, additional splicing rules, and gene architecture features to comprehensively evaluate the likelihood of a variant impacting splicing. Through extensive benchmarking across 21,000 splice-altering variants, Introme outperformed all tools (auPRC: 0.98) for the detection of clinically significant splice variants. Introme is available at https://github.com/CCICB/introme .Patricia J. Sullivan, Velimir Gayevskiy, Ryan L. Davis, Marie Wong, Chelsea Mayoh, Amali Mallawaarachchi, Yvonne Hort, Mark J. McCabe, Sarah Beecroft, Matilda R. Jackson, Peer Arts, Andrew Dubowsky, Nigel Laing, Marcel E. Dinger, Hamish S. Scott, Emily Oates, Mark Pinese, and Mark J. Cowle

Different types of disease-causing non-coding variants revealed by genomic and gene expression analyses in families with X-linked intellectual disability

The pioneering discovery research of X-linked intellectual disability (XLID) genes has benefitted thousands of individuals worldwide however, approximately 30% of XLID families still remain unresolved. We postulated that non-coding variants that affect gene regulation or splicing may account for the lack of a genetic diagnosis in some cases. Detecting pathogenic, gene-regulatory variants with the same sensitivity and specificity as structural and coding variants is a major challenge for Mendelian disorders. Here, we describe three pedigrees with suggestive XLID where distinctive phenotypes associated with known genes guided the identification of three different non-coding variants. We used comprehensive structural, single nucleotide and repeat expansion analyses of genome sequencing. RNA-Seq from patient-derived cell lines, RT-PCRs, western blots and reporter gene assays were used to confirm the functional effect of three fundamentally different classes of pathogenic non-coding variants: a retrotransposon insertion, a novel intronic splice donor and a canonical splice variant of an untranslated exon. In one family, we excluded a rare coding variant in ARX, a known XLID gene, in favour of a regulatory non-coding variant in OFD1 that correlated with the clinical phenotype. Our results underscore the value of genomic research on unresolved XLID families to aid novel, pathogenic non-coding variant discovery.Michael J. Field, Raman Kumar, Anna Hackett, Sayaka Kayumi, Cheryl A. Shoubridge, Lisa J. Ewans, Atma M. Ivancevic, Tracy Dudding, Byth, Renée Carroll, Thessa Kroes, Alison E. Gardner, Patricia Sullivan, Thuong T. Ha, Charles E. Schwartz, Mark J. Cowley, Marcel E. Dinger, Elizabeth E. Palmer, Louise Christie, Marie Shaw, Tony Roscioli, Jozef Gecz, Mark A. Corbet

A new class of non-coding RNAs associated with 3\u2019 untranslated regions of mRNAs

The importance of non-coding RNAs (ncRNAs) in controlling gene expression is becoming increasingly evident. However, except for some well characterized examples, such as miRNAs, Xist and Air, the function of most non-coding transcripts is still to be determined. Moreover, while small regulatory RNAs can be relatively easily classified on the basis of their length, secondary structure, and biochemical pathway, the classification of long \u201cmRNA-like\u201d ncRNAs has been problematic. Here we identify a large class of non-coding transcripts that originate within the 3\u2019UTR of at least one third of all genes in the mouse genome. We have several lines of evidence from genome-wide bioinformatic analyses (EST coverage, CAGE data, chromatin state maps of active promoters) and from invitro studies (strand-specific RT-PCR, 5\u2019RACE, Northern blot) showing that these 3\u2019UTR-associated ncRNAs (uaRNAs) can be either linked or transcribed separately to the upstream protein-coding sequences. In addition, expression profiles obtained by custom-designed microarrays on three different developmental systems (myoblast differentiation, male gonadal ridge formation, embryonic stem cell differentiation) showed that uaRNA expression is highly regulated and tissue-specific, and might be either concordant or discordant with respect to the upstream coding region depending on the cell type and on the developmental stage. This observation is confirmed by in-situ hybridization experiments, which evidenced that uaRNA and the associated coding transcript might have different subcellular locations. Our results highlight a further level of complexity at 3\u2019UTRs, suggesting the presence of new regulatory mechanisms that control gene expression during embryonic development. Our data have also important implications for the design of in-situ hybridization and microarray probes as well as for the interpretation of gene expression dat

Regulated independent expression of 3' untranslated regions in mammals

Eukaryotic mRNA localization, translation and stability are regulated by 3' untranslated regions (3'UTRs). 3'UTRs control mRNA expression in cis, via regulatory elements that are recognized by trans-acting factors, including RNA-binding proteins and microRNAs. However, a recent analysis of transcription start sites in mouse suggested that 3'UTRs may also be the source of independent transcripts, but there have been no in vivo analyses to confirm this suggestion. Here we report that a large number of 3'UTRs are not only linked to but are also expressed separately from their associated protein-coding sequences. We identify ~14,000 mouse genes exhibiting independent expression of 3'UTRs, of which 1,385 were supported by two distinct experimental approaches, and ~100 confirmed by microarray expression profiles. In situ hybridization demonstrated that 3'UTR-associated transcripts (uaRNA) can be expressed as part of mRNAs or discordantly in a developmentally-regulated and cell-specific manner, in some cases localizing to the nucleus. We also provide evidence that chromatin remodelling and transcription factor networks are involved in regulating uaRNA expression. These observations suggest that 3'UTRs not only function in cis to regulate protein expression but also in trans as noncoding RNAs, a conclusion supported by genetic studies dating back over a decade. Our findings prompt a re-evaluation of 3'UTR biology and the complexity of genome architecture in mammals

Aberrant RAG-mediated recombination contributes to multiple structural rearrangements in lymphoid blast crisis of chronic myeloid leukemia

Published: 19 February 2020Blast crisis of chronic myeloid leukemia is associated with poor survival and the accumulation of genomic lesions. Using whole-exome and/or RNA sequencing of patients at chronic phase (CP, n = 49), myeloid blast crisis (MBC, n = 19), and lymphoid blast crisis (LBC, n = 20), we found 25 focal gene deletions and 14 fusions in 24 patients in BC. Deletions predominated in LBC (83% of structural variants). Transcriptional analysis identified the upregulation of genes involved in V(D)J recombination, including RAG1/2 and DNTT in LBC. RAG recombination is a reported mediator of IKZF1 deletion. We investigated the extent of RAG-mediated genomic lesions in BC. Molecular hallmarks of RAG activity; DNTT-mediated nucleotide insertions and a RAG-binding motif at structural variants were exclusively found in patients with high RAG expression. Structural variants in 65% of patients in LBC displayed these hallmarks compared with only 5% in MBC. RAG-mediated events included focal deletion and novel fusion of genes associated with hematologic cancer: IKZF1, RUNX1, CDKN2A/B, and RB1. Importantly, 8/8 patients with elevated DNTT at CP diagnosis progressed to LBC by 12 months, potentially enabling early prediction of LBC. This work confirms the central mutagenic role of RAG in LBC and describes potential clinical utility in CML management.Daniel W. Thomson, Nur Hezrin Shahrin, Paul P. S. Wang, Carol Wadham, Naranie Shanmuganathan, Hamish S. Scott, Marcel E. Dinger, Timothy P. Hughes, Andreas W. Schreiber, Susan Branfor