16 results found
Beltran M, Yates CM, Skalska L, et al., 2016, The interaction of PRC2 with RNA or chromatin is mutually antagonistic, GENOME RESEARCH, Vol: 26, Pages: 896-907, ISSN: 1088-9051
Sibley CR, Blazquez L, Ule J, 2016, Lessons from non-canonical splicing, NATURE REVIEWS GENETICS, Vol: 17, Pages: 407-421, ISSN: 1471-0056
It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an 'RS-exon' that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5' splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5' splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons.
Wickramasinghe VO, Gonzàlez-Porta M, Perera D, et al., 2015, Regulation of constitutive and alternative mRNA splicing across the human transcriptome by PRPF8 is determined by 5' splice site strength., Genome Biol, Vol: 16
BACKGROUND: Sequential assembly of the human spliceosome on RNA transcripts regulates splicing across the human transcriptome. The core spliceosome component PRPF8 is essential for spliceosome assembly through its participation in ribonucleoprotein (RNP) complexes for splice-site recognition, branch-point formation and catalysis. PRPF8 deficiency is linked to human diseases like retinitis pigmentosa or myeloid neoplasia, but its genome-wide effects on constitutive and alternative splicing remain unclear. RESULTS: Here, we show that alterations in RNA splicing patterns across the human transcriptome that occur in conditions of restricted cellular PRPF8 abundance are defined by the altered splicing of introns with weak 5' splice sites. iCLIP of spliceosome components reveals that PRPF8 depletion decreases RNP complex formation at most splice sites in exon-intron junctions throughout the genome. However, impaired splicing affects only a subset of human transcripts, enriched for mitotic cell cycle factors, leading to mitotic arrest. Preferentially retained introns and differentially used exons in the affected genes contain weak 5' splice sites, but are otherwise indistinguishable from adjacent spliced introns. Experimental enhancement of splice-site strength in mini-gene constructs overcomes the effects of PRPF8 depletion on the kinetics and fidelity of splicing during transcription. CONCLUSIONS: Competition for PRPF8 availability alters the transcription-coupled splicing of RNAs in which weak 5' splice sites predominate, enabling diversification of human gene expression during biological processes like mitosis. Our findings exemplify the regulatory potential of changes in the core spliceosome machinery, which may be relevant to slow-onset human genetic diseases linked to PRPF8 deficiency.
Huppertz I, Attig J, D'Ambrogio A, et al., 2014, iCLIP: protein-RNA interactions at nucleotide resolution., Methods, Vol: 65, Pages: 274-287
RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. The high resolution and specificity of this method are achieved by an intramolecular cDNA circularization step that enables analysis of cDNAs that truncated at the protein-RNA crosslink sites. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs.
Sibley CR, 2014, Regulation of gene expression through production of unstable mRNA isoforms., Biochem Soc Trans, Vol: 42, Pages: 1196-1205
Alternative splicing is universally accredited for expanding the information encoded within the transcriptome. In recent years, several tightly regulated alternative splicing events have been reported which do not lead to generation of protein products, but lead to unstable mRNA isoforms. Instead these transcripts are targets for NMD (nonsense-mediated decay) or retained in the nucleus and degraded. In the present review I discuss the regulation of these events, and how many have been implicated in control of gene expression that is instrumental to a number of developmental paradigms. I further discuss their relevance to disease settings and conclude by highlighting technologies that will aid identification of more candidate events in future.
Modic M, Ule J, Sibley CR, 2013, CLIPing the brain: studies of protein-RNA interactions important for neurodegenerative disorders., Mol Cell Neurosci, Vol: 56, Pages: 429-435
The fate of an mRNA is largely determined by its interactions with RNA binding proteins (RBPs). Post-transcriptional processing, RNA stability, localisation and translation are some of the events regulated by the plethora of RBPs present within cells. Mutations in various RBPs cause several diseases of the central nervous system, including frontotemporal lobar degeneration, amyotrophic lateral sclerosis and fragile X syndrome. Here we review the studies that integrated UV-induced cross-linked immunoprecipitation (CLIP) with other genome-wide methods to comprehensively characterise the function of diverse RBPs in the brain. We discuss the technical challenges of these studies and review the strategies that can be used to reliably identify the RNAs bound and regulated by an RBP. We conclude by highlighting how CLIP and related techniques have been instrumental in addressing the role of RBPs in neurologic diseases. This article is part of a Special Issue entitled: RNA and splicing regulation in neurodegeneration.
Curtis HJ, Sibley CR, Wood MJ, 2012, Mirtrons, an emerging class of atypical miRNA., Wiley Interdiscip Rev RNA, Vol: 3, Pages: 617-632
Post-transcriptional gene silencing (PTGS) via RNA interference (RNAi) is a vital gene regulatory mechanism for fine-tuning gene expression. RNAi effectors termed microRNAs (miRNAs) are implicated in various aspects of animal development and normal physiological function, while dysregulation has been linked to several pathologies. Several atypical miRNA biogenesis pathways have been identified, yet in most cases the reasons for their emergence remain unclear. One of these atypical pathways is the mirtron pathway, where short introns are excised by splicing to generate intermediates of the RNAi pathway, with no cleavage by the microprocessor. Closely related pathways involving tailed-mirtron and simtron biogenesis have also been described. There is extensive evidence that mirtrons function as miRNAs, and while some are evolutionarily conserved across similar species, others appear to have emerged relatively recently. In addition, through exploitation of the potent and sequence-specific silencing capabilities of RNAi, synthetic mirtrons may have potential for overcoming certain therapeutic challenges.
Patani R, Sibley CR, Chandran S, et al., 2012, Using human pluripotent stem cells to study post-transcriptional mechanisms of neurodegenerative diseases., Brain Res, Vol: 1462, Pages: 129-138
Post-transcriptional regulation plays a major role in the generation of cell type diversity. In particular, alternative splicing increases diversification of transcriptome between tissues, in different cell types within a tissue, and even in different compartments of the same cell. The complexity of alternative splicing has increased during evolution. With increasing sophistication, however, comes greater potential for malfunction of these intricate processes. Indeed, recent years have uncovered a wealth of disease-causing mutations affecting RNA-binding proteins and non-coding regions on RNAs, highlighting the importance of studying disease mechanisms that act at the level of RNA processing. For instance, mutations in TARDBP and FUS, or a repeat expansion in the intronic region of the C9ORF72 gene, can all cause amyotrophic lateral sclerosis. We discuss how interspecies differences highlight the necessity for human model systems to complement existing non-human approaches to study neurodegenerative disorders. We conclude by discussing the improvements that could further increase the promise of human pluripotent stem for cell-based disease modeling. This article is part of a Special Issue entitled "RNA-Binding Proteins".
Seow Y, Sibley CR, Wood MJ, 2012, Artificial mirtron-mediated gene knockdown: functional DMPK silencing in mammalian cells., RNA, Vol: 18, Pages: 1328-1337
Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs. Here we present a design algorithm for artificial mirtrons and demonstrate, for the first time, efficient gene knockdown of myotonic dystrophy protein kinase (DMPK) target sequences in Renilla luciferase 3' UTR and subsequently pathogenic DMPK mRNA, causative of Type I myotonic dystrophy, using artificial mirtrons cloned as eGFP introns. Deep sequencing of artificial mirtrons suggests that functional mature transcripts corresponding to the designed sequence were produced in high abundance. They were further shown to be splicing-dependent, Drosha-independent, and partially dependent on exportin-5, resulting in the precise generation of pre-miRNAs. In a murine myoblast line containing a pathogenic copy of human DMPK with more than 500 CUG repeats, the DMPK artificial mirtron corrected DM1-associated splicing abnormalities of the Serca-1 mRNA, demonstrating the therapeutic potential of mirtron-mediated RNAi. Thus, further development and exploitation of the unique properties of mirtrons will benefit future research and therapeutic RNAi applications as an alternative to conventional RNAi strategies.
Sibley CR, Attig J, Ule J, 2012, The greatest catch: big game fishing for mRNA-bound proteins, Genome Biology, Vol: 13, Pages: 163-163, ISSN: 1465-6906
Sibley CR, Seow Y, Curtis H, et al., 2012, Silencing of Parkinson's disease-associated genes with artificial mirtron mimics of miR-1224., Nucleic Acids Res, Vol: 40, Pages: 9863-9875
Mirtrons are a recently described category of microRNA (miRNA) relying on splicing rather than processing by the microprocessor complex to generate pre-miRNA precursors of the RNA interference (RNAi) pathway. Their discovery and subsequent verification provides important information about a distinct class of miRNA and inherent advantages that could be exploited to silence genes of interest. These include micro-processor-independent biogenesis, pol-II-dependent transcription, accurate species generation and the delivery of multiple artificial mirtrons as introns within a single host transcript. Here we determined the sequence motifs required for correct processing of the mmu-miR-1224 mirtron and incorporated these into artificial mirtrons targeting Parkinson's disease-associated LRRK2 and α-synuclein genes. By incorporating these rules associated with processing and splicing, artificial mirtrons could be designed and made to silence complementary targets either at the mRNA or protein level. We further demonstrate with a LRRK2 targeting artificial mirtron that neuronal-specific silencing can be directed under the control of the human synapsin promoter. Finally, multiple mirtrons were co-delivered within a single host transcript, an eGFP reporter, to allow simultaneous targeting of two or more targets in a combinatorial approach. Thus, the unique characteristics of artificial mirtrons make this an attractive approach for future RNAi applications.
Sibley CR, Seow Y, Saayman S, et al., 2012, The biogenesis and characterization of mammalian microRNAs of mirtron origin., Nucleic Acids Res, Vol: 40, Pages: 438-448
Mirtrons, short hairpin pre-microRNA (miRNA) mimics directly produced by intronic splicing, have recently been identified and experimentally confirmed in invertebrates. While there is evidence to suggest several mammalian miRNAs have mirtron origins, this has yet to be experimentally demonstrated. Here, we characterize the biogenesis of mammalian mirtrons by ectopic expression of splicing-dependent mirtron precursors. The putative mirtrons hsa-miR-877, hsa-miR-1226 and mmu-miR-1224 were designed as introns within eGFP. Correct splicing and function of these sequences as introns was shown through eGFP fluorescence and RT-PCR, while all mirtrons suppressed perfectly complementary luciferase reporter targets to levels similar to that of corresponding independently expressed pre-miRNA controls. Splicing-deficient mutants and disruption of key steps in miRNA biogenesis demonstrated that mirtron-mediated gene knockdown was splicing-dependent, Drosha-independent and had variable dependence on RNAi pathway elements following pre-miRNA formation. The silencing effect of hsa-miR-877 was further demonstrated to be mediated by the generation of short anti-sense RNA species expressed with low abundance. Finally, the mammalian mirtron hsa-miR-877 was shown to reduce mRNA levels of an endogenous transcript containing hsa-miR-877 target sites in neuronal SH-SY5Y cells. This work confirms the mirtron origins of three mammalian miRNAs and suggests that they are a functional class of splicing-dependent miRNAs which are physiologically active.
Sibley CR, Wood MJ, 2011, Identification of allele-specific RNAi effectors targeting genetic forms of Parkinson's disease., PLoS One, Vol: 6
Parkinson's disease (PD) is a progressive neurological disorder affecting an estimated 5-10 million people worldwide. Recent evidence has implicated several genes that directly cause or increase susceptibility to PD. As well as advancing understanding of the genetic aetiology of PD these findings suggest new ways to modify the disease course, in some cases through genetic manipulation. Here we generated a 'walk-through' series of RNA Pol III-expressed shRNAs targeting both the α-synuclein A30P and LRRK2 G2019S PD-associated mutations. Allele-specific discrimination of the α-synuclein A30P mutation was achieved with alignments at position 10, 13 and 14 in two model systems, including a heterozygous model mimicking the disease setting, whilst 5'RACE was used to confirm stated alignments. Discrimination of the most common PD-linked LRRK2 G2019S mutation was assessed in hemizygous dual-luciferase assays and showed that alignment of the mutation opposite position 4 of the antisense species produced robust discrimination of alleles at all time points studied. Discrimination at this position was subsequently confirmed using siRNAs, where up to 10-fold discrimination was seen. The results suggest that RNAi-mediated silencing of PD-associated autosomal dominant genes could be a novel therapeutic approach for the treatment of the relevant clinical cases of PD in future.
Sibley CR, Wood MJ, 2011, The miRNA pathway in neurological and skeletal muscle disease: implications for pathogenesis and therapy., J Mol Med (Berl), Vol: 89, Pages: 1065-1077
RNA interference (RNAi) represents a powerful post-transcriptional gene silencing network which fine-tunes gene expression in all eukaryotic cells. The endogenous triggers of RNAi, microRNAs (miRNAs), are proposed to regulate expression of up to a third of all protein-coding genes, and have been shown to have critical roles in developmental processes including in the central nervous system and skeletal muscle. Further, many have been reported to display differential expression in various disease states. Here we describe present understanding of the biogenesis and function of miRNAs, review current knowledge of miRNA abnormalities in both human neurological and skeletal muscle disease and discuss their potential as novel disease biomarkers. Finally, we highlight the many ways in which the miRNA pathway may be targeted for therapeutic benefit.
Sibley CR, Seow Y, Wood MJ, 2010, Novel RNA-based strategies for therapeutic gene silencing., Mol Ther, Vol: 18, Pages: 466-476
The past decade has seen intense scientific interest in non-coding RNAs. In particular, the discovery and subsequent exploitation of gene silencing via RNA interference (RNAi) has revolutionized the way in which gene expression is now studied and understood. It is now well established that post-transcriptional gene silencing (PTGS) by the microRNA (miRNA) and other RNAi-associated pathways represents an essential layer of complexity to gene regulation. Gene silencing using RNAi additionally demonstrates huge potential as a therapeutic strategy for eliminating pathogenic gene expression. Yet despite the early promise and excitement of gene-specific silencing, several critical hurdles remain to be overcome before widespread clinical adoption. These include off-target effects, toxicity due to saturation of the endogenous RNAi functions, limited duration of silencing, and effective targeted delivery. In recent years, a range of novel strategies for producing RNA-mediated silencing have been developed that can circumvent many of these hurdles, including small internally segmented interfering RNAs, tandem hairpin RNAs, and pri-miRNA cluster mimics. This review discusses RNA-mediated silencing in light of this recent research, and highlights the benefits and limitations conferred by these novel gene-silencing strategies.
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