Ranking noncanonical 5′ splice site usage by genome-wide RNA-seq analysis and splicing reporter assays

AbstractMost protein encoding genes in eukaryotes contain introns which are interwoven with exons. After transcription, introns need to be removed in order to generate the final mRNA which can be translated into an amino acid sequence. Precise excision of introns by the spliceosome requires conserved dinucleotides which mark the splice sites. However, there are variations of the highly conserved combination of GT at the 5’ end and AG at the 3’ end of an intron in the genome. GC-AG and AT-AC are two major non-canonical splice site combinations which have been known for years. During the last years, various minor non-canonical splice site combinations were detected with numerous dinucleotide permutations. Here we expand systematic investigations of non-canonical splice site combinations in plants to all eukaryotes by analysing fungal and animal genome sequences. Comparisons of splice site combinations between these three kingdoms revealed several differences such as a substantially increased CT-AC frequency in fungal genome sequences. Canonical GT-AG splice site combinations in antisense transcripts could be one explanation for this observation. In addition, high numbers of GA-AG splice site combinations were observed in Eurytemora affinis and Oikopleura dioica. A variant in one U1 snRNA isoform might allow the recognition of GA as 5’ splice site. In depth investigation of splice site usage based on RNA-Seq read mappings indicates a generally higher flexibility of the 3’ splice site compared to the 5’ splice site across animals, fungi, and plants.

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Interrogation of genome-wide, experimentally dissected gene regulatory networks reveals mechanisms underlying dynamic cellular state control

10.1101/2021.06.28.449297 ◽

2021 ◽

Author(s):

Xiangtian Tan ◽

Jeremy Worley ◽

Mikko Turunen ◽

Kelly Wong ◽

Ester Calvo Fernandez ◽

...

Keyword(s):

Regulatory Networks ◽

Regulatory Protein ◽

State Control ◽

Rna Seq ◽

Protein Activity ◽

Genome Wide ◽

Adenocarcinoma Cells ◽

Reporter Assays ◽

Gene Regulatory ◽

Context Specific

Pooled CRISPRi-mediated silencing of >1,000 transcriptional regulators expressed in single colorectal adenocarcinoma cells, followed by single-cell RNA-seq profiling at two time points, 1D and 4D, allowed reverse engineering the underlying tumor context-specific, causal regulatory network. Furthermore, the availability of experimentally derived, highly multiplexed gene reporter assays for each regulator, as identified by this analysis, allowed accurate assessment of differential protein activity following silencing of each regulator, thus providing proof-of-concept for generating comprehensive, tissue-specific networks of transcriptional and post-translational interactions. Analysis of this causal network allowed elucidation of complex autoregulatory mechanisms that have eluded previous computational approaches and supported systematic elucidation of cooperative mechanisms, where one regulatory protein can modulate the activity of another regulatory protein, as well as transcriptional mimicry, where one regulatory protein can phenocopy others.

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Exploration of the cyanidioschyzon merolae intron landscape

10.24124/2021/59202 ◽

2021 ◽

Author(s):

◽

Viktor Slat

Keyword(s):

Genome Engineering ◽

Cyanidioschyzon Merolae ◽

Rna Seq ◽

Protein Coding ◽

Genome Wide ◽

Accurate Picture ◽

Small Nuclear Rnas ◽

Associated Proteins ◽

Splice Site Usage

The eukaryotic process of pre-mRNA splicing involves the removal of noncoding intron sequences and the fusion of the remaining protein-coding exon sequences. The splicing reaction is catalyzed by the spliceosome, a dynamic multi-megadalton ribonucleoprotein complex that, in humans, is composed of 5 small nuclear RNAs (snRNAs) and over 200 associated proteins acting on more that 200,000 introns present within 25,000 genes. The unicellular red alga Cyanidioschyzon merolae possesses a more tractable splicing environment, with only 4 snRNAs and 75 associated proteins interacting with 27 annotated introns found in 26 our of 5,331 genes. Intron-rich genomes can confer benefits to their host species such as improved gene expression, incredible proteomic diversity, and increased genetic stability. This raises the question of why intron-poor C. merolae has retained such a small number of introns and a dramatically reduced spliceosome. A comprehensive investigation into the precise role that introns play in C. merolae would require the systematic removal of introns and an analysis of the effects thereof. The ability to elucidate the role of splicing in C. merolae via genome-wide intron deletion, however, hinges on the feasibility of establishing the efficiently scalable CRISPR genome engineering tool in C. merolae. It also follows that such an endeavour would require an accurate picture of the intron landscape of C. merolae, and since the number of annotated introns in C. merolae is relatively small, it is especially vital to determine whether any introns are missing from the C. merolae annotation. To that end, a stable and inducible Cas9-expressing strain of C. merolae was successfully developed. Transcriptome analysis using RNA-seq data revealed the discovery of 11 novel introns and 1 misannotated intron, as well as the presence of alternative splicing in the form of alternative splice site usage.

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An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys

10.1101/258897 ◽

2018 ◽

Author(s):

Marilyn Parra ◽

Ben W. Booth ◽

Richard Weiszmann ◽

Brian Yee ◽

Gene W. Yeo ◽

...

Keyword(s):

Intron Retention ◽

Developmental Regulation ◽

Splice Junction ◽

Rna Seq ◽

Nonsense Mediated Decay ◽

Genome Wide ◽

Reporter Assays ◽

Retained Introns ◽

Splice Junctions ◽

Intron 4

AbstractDuring terminal erythropoiesis, the splicing machinery in differentiating erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ~50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron. Minigene splicing reporter assays showed that these cassettes promote IR. Genome-wide analysis of splice junction reads demonstrated that cryptic noncoding cassettes are much more common in large (>1kb) retained introns than they are in small retained introns or in non-retained introns. Functional assays showed that heterologous cassettes can promote retention of intron 4 in the SF3B1 splicing reporter. Although many of these cryptic exons were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that these exons function as decoys that engage the intron-terminal splice sites, blocking cross-intron interactions required for excision. Developmental regulation of decoy function underlies a major component of the erythroblast IR program.

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VELCRO-IP RNA-seq reveals ribosome expansion segment function in translation genome-wide

Cell Reports ◽

10.1016/j.celrep.2020.108629 ◽

2021 ◽

Vol 34 (3) ◽

pp. 108629

Author(s):

Kathrin Leppek ◽

Gun Woo Byeon ◽

Kotaro Fujii ◽

Maria Barna

Keyword(s):

Rna Seq ◽

Expansion Segment ◽

Genome Wide

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Distinct regulation of hippocampal neuroplasticity and ciliary genes by corticosteroid receptors

Nature Communications ◽

10.1038/s41467-021-24967-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Karen R. Mifsud ◽

Clare L. M. Kennedy ◽

Silvia Salatino ◽

Eshita Sharma ◽

Emily M. Price ◽

...

Keyword(s):

Dna Sequences ◽

Glucocorticoid Receptors ◽

Acute Stress ◽

Circadian Variation ◽

Rna Seq ◽

Physiological Regulation ◽

Behavioural Adaptation ◽

Neuronal Progenitor ◽

Genome Wide ◽

Transcriptional Changes

AbstractGlucocorticoid hormones (GCs) — acting through hippocampal mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) — are critical to physiological regulation and behavioural adaptation. We conducted genome-wide MR and GR ChIP-seq and Ribo-Zero RNA-seq studies on rat hippocampus to elucidate MR- and GR-regulated genes under circadian variation or acute stress. In a subset of genes, these physiological conditions resulted in enhanced MR and/or GR binding to DNA sequences and associated transcriptional changes. Binding of MR at a substantial number of sites however remained unchanged. MR and GR binding occur at overlapping as well as distinct loci. Moreover, although the GC response element (GRE) was the predominant motif, the transcription factor recognition site composition within MR and GR binding peaks show marked differences. Pathway analysis uncovered that MR and GR regulate a substantial number of genes involved in synaptic/neuro-plasticity, cell morphology and development, behavior, and neuropsychiatric disorders. We find that MR, not GR, is the predominant receptor binding to >50 ciliary genes; and that MR function is linked to neuronal differentiation and ciliogenesis in human fetal neuronal progenitor cells. These results show that hippocampal MRs and GRs constitutively and dynamically regulate genomic activities underpinning neuronal plasticity and behavioral adaptation to changing environments.

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SPLICE-q: a Python tool for genome-wide quantification of splicing efficiency

BMC Bioinformatics ◽

10.1186/s12859-021-04282-6 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Verônica R. de Melo Costa ◽

Julianus Pfeuffer ◽

Annita Louloupi ◽

Ulf A. V. Ørom ◽

Rosario M. Piro

Keyword(s):

Gene Expression ◽

Cancer Progression ◽

Time Course ◽

Progressive Increase ◽

Rna Seq ◽

Transcript Processing ◽

Aberrant Transcript ◽

Genome Wide ◽

Splicing Efficiency ◽

Nascent Rna

Abstract Background Introns are generally removed from primary transcripts to form mature RNA molecules in a post-transcriptional process called splicing. An efficient splicing of primary transcripts is an essential step in gene expression and its misregulation is related to numerous human diseases. Thus, to better understand the dynamics of this process and the perturbations that might be caused by aberrant transcript processing it is important to quantify splicing efficiency. Results Here, we introduce SPLICE-q, a fast and user-friendly Python tool for genome-wide SPLICing Efficiency quantification. It supports studies focusing on the implications of splicing efficiency in transcript processing dynamics. SPLICE-q uses aligned reads from strand-specific RNA-seq to quantify splicing efficiency for each intron individually and allows the user to select different levels of restrictiveness concerning the introns’ overlap with other genomic elements such as exons of other genes. We applied SPLICE-q to globally assess the dynamics of intron excision in yeast and human nascent RNA-seq. We also show its application using total RNA-seq from a patient-matched prostate cancer sample. Conclusions Our analyses illustrate that SPLICE-q is suitable to detect a progressive increase of splicing efficiency throughout a time course of nascent RNA-seq and it might be useful when it comes to understanding cancer progression beyond mere gene expression levels. SPLICE-q is available at: https://github.com/vrmelo/SPLICE-q

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Genome-Wide Identification and Characterization of Hsf and Hsp Gene Families and Gene Expression Analysis under Heat Stress in Eggplant (Solanum melongema L.)

Horticulturae ◽

10.3390/horticulturae7060149 ◽

2021 ◽

Vol 7 (6) ◽

pp. 149

Author(s):

Chao Gong ◽

Qiangqiang Pang ◽

Zhiliang Li ◽

Zhenxing Li ◽

Riyuan Chen ◽

...

Keyword(s):

Heat Stress ◽

Gene Families ◽

High Temperature Stress ◽

Pcr Analysis ◽

Rna Seq ◽

Genome Wide ◽

Motif Composition ◽

Hsp Genes ◽

Identification And Characterization

Under high temperature stress, a large number of proteins in plant cells will be denatured and inactivated. Meanwhile Hsfs and Hsps will be quickly induced to remove denatured proteins, so as to avoid programmed cell death, thus enhancing the thermotolerance of plants. Here, a comprehensive identification and analysis of the Hsf and Hsp gene families in eggplant under heat stress was performed. A total of 24 Hsf-like genes and 117 Hsp-like genes were identified from the eggplant genome using the interolog from Arabidopsis. The gene structure and motif composition of Hsf and Hsp genes were relatively conserved in each subfamily in eggplant. RNA-seq data and qRT-PCR analysis showed that the expressions of most eggplant Hsf and Hsp genes were increased upon exposure to heat stress, especially in thermotolerant line. The comprehensive analysis indicated that different sets of SmHsps genes were involved downstream of particular SmHsfs genes. These results provided a basis for revealing the roles of SmHsps and SmHsp for thermotolerance in eggplant, which may potentially be useful for understanding the thermotolerance mechanism involving SmHsps and SmHsp in eggplant.

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FOXO1 mitigates the SMAD3/FOXL2C134W transcriptomic effect in a model of human adult granulosa cell tumor

Journal of Translational Medicine ◽

10.1186/s12967-021-02754-0 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Christian Secchi ◽

Paola Benaglio ◽

Francesca Mulas ◽

Martina Belli ◽

Dwayne Stupack ◽

...

Keyword(s):

Granulosa Cell ◽

Chromatin Remodeling ◽

Granulosa Cell Tumor ◽

Cell Tumor ◽

Rna Seq ◽

Pathogenic Role ◽

Rare Type ◽

Cellular Models ◽

Genome Wide

Abstract Background Adult granulosa cell tumor (aGCT) is a rare type of stromal cell malignant cancer of the ovary characterized by elevated estrogen levels. aGCTs ubiquitously harbor a somatic mutation in FOXL2 gene, Cys134Trp (c.402C < G); however, the general molecular effect of this mutation and its putative pathogenic role in aGCT tumorigenesis is not completely understood. We previously studied the role of FOXL2C134W, its partner SMAD3 and its antagonist FOXO1 in cellular models of aGCT. Methods In this work, seeking more comprehensive profiling of FOXL2C134W transcriptomic effects, we performed an RNA-seq analysis comparing the effect of FOXL2WT/SMAD3 and FOXL2C134W/SMAD3 overexpression in an established human GC line (HGrC1), which is not luteinized, and bears normal alleles of FOXL2. Results Our data shows that FOXL2C134W/SMAD3 overexpression alters the expression of 717 genes. These genes include known and novel FOXL2 targets (TGFB2, SMARCA4, HSPG2, MKI67, NFKBIA) and are enriched for neoplastic pathways (Proteoglycans in Cancer, Chromatin remodeling, Apoptosis, Tissue Morphogenesis, Tyrosine Kinase Receptors). We additionally expressed the FOXL2 antagonistic Forkhead protein, FOXO1. Surprisingly, overexpression of FOXO1 mitigated 40% of the altered genome-wide effects specifically related to FOXL2C134W, suggesting it can be a new target for aGCT treatment. Conclusions Our transcriptomic data provide novel insights into potential genes (FOXO1 regulated) that could be used as biomarkers of efficacy in aGCT patients.

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Extremes of age are associated with differences in the expression of selected pattern recognition receptor genes and ACE2, the receptor for SARS-CoV-2: implications for the epidemiology of COVID-19 disease

BMC Medical Genomics ◽

10.1186/s12920-021-00970-7 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Stephen W. Bickler ◽

David M. Cauvi ◽

Kathleen M. Fisch ◽

James M. Prieto ◽

Alicia G. Sykes ◽

...

Keyword(s):

Pattern Recognition ◽

Dermal Fibroblasts ◽

Pattern Recognition Receptor ◽

Human Dermal Fibroblasts ◽

Rna Seq ◽

Large Dataset ◽

Genome Wide ◽

The Individual ◽

Recognition Receptor

Abstract Background Older aged adults and those with pre-existing conditions are at highest risk for severe COVID-19 associated outcomes. Methods Using a large dataset of genome-wide RNA-seq profiles derived from human dermal fibroblasts (GSE113957) we investigated whether age affects the expression of pattern recognition receptor (PRR) genes and ACE2, the receptor for SARS-CoV-2. Results Extremes of age are associated with increased expression of selected PRR genes, ACE2 and four genes that encode proteins that have been shown to interact with SAR2-CoV-2 proteins. Conclusions Assessment of PRR expression might provide a strategy for stratifying the risk of severe COVID-19 disease at both the individual and population levels.

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