Detection of generic differential RNA processing events from RNA-seq data

Somatic cell nuclear transfer (SCNT) is not only a valuable tool for understanding nuclear reprogramming, but it also facilitates the generation of genetically modified animals. However, the development of SCNT embryos has remained an uncontrollable process. It was reported that the SCNT embryos that complete the first cell division sooner are more likely to develop to the blastocyst stage, suggesting their better developmental competence. Therefore, to better understand the underlying molecular mechanisms, RNA-seq of pig SCNT embryos that were early-dividing (24 h postactivation) and late-dividing (36 h postactivation) was performed. Our analysis revealed that early- and late-dividing embryos have distinct RNA profiles, and, in all, 3077 genes were differentially expressed. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that early-dividing embryos exhibited higher expression in genes that participated in the meiotic cell cycle, while enrichment of RNA processing- and translation-related genes was found in late-dividing embryos. There are also fewer somatic memory genes such as FLRT2, ADAMTS1, and FOXR1, which are abnormally activated or suppressed in early-dividing cloned embryos. These results show that early-dividing SCNT embryos have different transcriptional profiles than late-dividing embryos. Early division of SCNT embryos may be associated with their better reprogramming capacity, and somatic memory genes may act as a reprogramming barrier in pig SCNT reprogramming.

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RJunBase: a database of RNA splice junctions in human normal and cancerous tissues

Nucleic Acids Research ◽

10.1093/nar/gkaa1056 ◽

2020 ◽

Vol 49 (D1) ◽

pp. D201-D211

Author(s):

Qin Li ◽

Hongyan Lai ◽

Yuchen Li ◽

Bing Chen ◽

Siyuan Chen ◽

...

Keyword(s):

Cancer Treatment ◽

Expression Profile ◽

Rna Processing ◽

Human Cells ◽

Rna Seq ◽

Essential Step ◽

Splicing Pattern ◽

Gene Level ◽

Precursor Rna ◽

Splice Junctions

Abstract Splicing is an essential step of RNA processing for multi-exon genes, in which introns are removed from a precursor RNA, thereby producing mature RNAs containing splice junctions. Here, we develope the RJunBase (www.RJunBase.org), a web-accessible database of three types of RNA splice junctions (linear, back-splice, and fusion junctions) that are derived from RNA-seq data of non-cancerous and cancerous tissues. The RJunBase aims to integrate and characterize all RNA splice junctions of both healthy or pathological human cells and tissues. This new database facilitates the visualization of the gene-level splicing pattern and the junction-level expression profile, as well as the demonstration of unannotated and tumor-specific junctions. The first release of RJunBase contains 682 017 linear junctions, 225 949 back-splice junctions and 34 733 fusion junctions across 18 084 non-cancerous and 11 540 cancerous samples. RJunBase can aid researchers in discovering new splicing-associated targets and provide insights into the identification and assessment of potential neoepitopes for cancer treatment.

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RNA-Seq Analysis of lncRNAs and cisNATs in the Tomato Genome

IU Journal of Undergraduate Research ◽

10.14434/iujur.v1i1.13244 ◽

2015 ◽

Vol 1 (1) ◽

pp. 34

Author(s):

S. Hussain Ather

Keyword(s):

Rna Processing ◽

De Novo ◽

Rna Seq ◽

Tomato Genome ◽

Biological Mechanisms ◽

Global Issues ◽

Tomato Species ◽

And Function ◽

Two Stages ◽

Coding Potential

Since the sequencing of the human genome, it has been revealed that the vast majority of DNA does not code for proteins. Instead, these regions of DNA produce long noncoding RNAs (lncRNAs), which have recently been reported to play important roles such as protein regulation and small RNA processing (Wilusz, Sunwoo, & Spector, 2009). The catalog and functions of lncRNAs in the ripening of tomato species (Solanum lycopersicum) are largely unknown. Similarly, the mechanisms of cis-natural antisense transcripts (cisNATs) of proximal complementary RNA strings, which function to inhibit transcription, are also poorly understood (Wang, Gaasterland, & Chua, 2005). Global issues in food production and malnutrition exacerbate the relevance of understanding these biological mechanisms central to the development of fruit. We identified certain functions of lncRNAs and cisNATs in the tomato ripening process using an RNA-Seq pipeline (Wang, Gerstein, & Snyder, 2005). Raw reads from two different stages in the tomato ripening cycle were aligned to a reference genome to test the hypothesis that there would be different expression levels for certain lncRNAs and cis-NATs between the two stages. The two stages were Mature Green, the stage in which the tomato is completely green, and Breaker, the stage in which the tomato shows initial colors of red. Then, the reads were de novo assembled, assessed for coding potential, and annotated by transcript and function. Finally, the results were filtered for lncRNAs (length > 200 bp, ORF < 100 bp, noncoding, expression value > 0) and cis-NATs (sense-antisense pairs, overlap length > 50 bp, differential splice patterns, expression value = 0). Differentially-expressed lncRNAs and cis-NATs between the two stages of development were identified, and their functions were analyzed. However, experimental evidence is necessary to confirm our findings and hypothesize models of cis-NAT mechanisms for further classification and identification.

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Transcriptome-wide splicing quantification in single cells

10.1101/098517 ◽

2017 ◽

Cited By ~ 1

Author(s):

Yuanhua Huang ◽

Guido Sanguinetti

Keyword(s):

Rna Processing ◽

Single Cells ◽

Bayesian Hierarchical Model ◽

Bayesian Regression ◽

Data Sets ◽

Rna Seq ◽

Bayesian Hierarchical ◽

Informative Prior Distribution ◽

Comprehensive Measurement ◽

Isoform Quantification

AbstractSingle cell RNA-seq (scRNA-seq) has revolutionised our understanding of transcriptome variability, with profound implications both fundamental and translational. While scRNA-seq provides a comprehensive measurement of stochasticity in transcription, the limitations of the technology have prevented its application to dissect variability in RNA processing events such as splicing. Here we present BRIE (Bayesian Regression for Isoform Estimation), a Bayesian hierarchical model which resolves these problems by learning an informative prior distribution from sequence features. We show that BRIE yields reproducible estimates of exon inclusion ratios in single cells and provides an effective tool for differential isoform quantification between scRNA-seq data sets. BRIE therefore expands the scope of scRNA-seq experiments to probe the stochasticity of RNA-processing.

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Metabolic labeling of RNAs uncovers hidden features and dynamics of the Arabidopsis thaliana transcriptome

10.1101/588780 ◽

2019 ◽

Author(s):

Emese Xochitl Szabo ◽

Philipp Reichert ◽

Marie-Kristin Lehniger ◽

Marilena Ohmer ◽

Marcella de Francisco Amorim ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Transcriptome Analysis ◽

Rna Processing ◽

Rna Synthesis ◽

Rna Stability ◽

Metabolic Labeling ◽

Rna Seq ◽

Genome Wide ◽

Stability Measurement ◽

Chemical Inhibition

AbstractTranscriptome analysis by RNA sequencing (RNA-seq) has become an indispensable core research tool in modern plant biology. Virtually all RNA-seq studies provide a snapshot of the steady-state transcriptome, which contains valuable information about RNA populations at a given time, but lacks information about the dynamics of RNA synthesis and degradation. Only a few specialized sequencing techniques, such as global run-on sequencing (GRO-seq), have been applied in plants and provide information about RNA synthesis rates. Here, we demonstrate that RNA labeling with a modified, non-toxic uridine analog, 5-ethynyl uridine (5-EU), in Arabidopsis thaliana seedlings provides insight into the dynamic nature of a plant transcriptome. Pulse-labeling with 5-EU allowed the detection and analysis of nascent and unstable RNAs, of RNA processing intermediates generated by splicing, and of chloroplast RNAs. We also conducted pulse-chase experiments with 5-EU, which allowed us to determine RNA stabilities without the need for chemical inhibition of transcription using compounds such as actinomycin and cordycepin. Genome-wide analysis of RNA stabilities by 5-EU pulse-chase experiments revealed that this inhibitor-free RNA stability measurement results in RNA half-lives much shorter than those reported after chemical inhibition of transcription. In summary, our results show that the Arabidopsis nascent transcriptome contains unstable RNAs and RNA processing intermediates, and suggest that half-lives of plant RNAs are largely overestimated. Our results lay the ground for an easy and affordable nascent transcriptome analysis and inhibitor-free analysis of RNA stabilities in plants.

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Frontiers in RNA biology: advances from a small fly Drosophila melanogaster

The Biochemist ◽

10.1042/bio03802021 ◽

2016 ◽

Vol 38 (2) ◽

pp. 21-25

Author(s):

James B. Brown ◽

Susan E. Celniker

Keyword(s):

Rna Processing ◽

Small Sample ◽

Rna Seq ◽

Ribonucleoprotein Complexes ◽

Rna Biology ◽

Cell Type Specific ◽

Exciting Time ◽

Small Sample Sizes ◽

Analytical Tools ◽

Processing Mechanisms

In this article, we discuss emerging frontiers in RNA biology from a historical perspective. The field is currently undergoing yet another transformative expansion. RNA-seq has revealed that splicing, and, more generally, RNA processing is far more complex than expected, and the mechanisms of regulation are correspondingly sophisticated. Our understanding of the molecular machines involved in RNA metabolism is incomplete and derives from small sample sizes. Even if we manage to complete a catalogue of molecular species, RNA isoforms and the ribonucleoprotein complexes that drive their genesis, the horizons of molecular dynamics and cell-type-specific processing mechanisms await. This is an exciting time to enter into the study of RNA biology; analytical tools, wet and dry, are advancing rapidly, and each new measurement modality brings into view another new function or activity of versatile RNA. Since the dawn of sequence-based RNA biology, we have come a long way.

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The Alternative Splicing Landscape in Multiple Myeloma Is Determined By IGH Translocations and Mutations of RNA Processing Genes

Blood ◽

10.1182/blood-2021-153113 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2689-2689

Author(s):

Enze Liu ◽

Sabine Wenzel ◽

Brian A Walker

Keyword(s):

Multiple Myeloma ◽

Survival Analysis ◽

Alternative Splicing ◽

High Risk ◽

Cell Growth ◽

Rna Processing ◽

Cox Regression ◽

P Value ◽

Rna Seq ◽

Logrank Test

Abstract Introduction: Alternative Splicing (AS) plays a key role in regulating numerous cellular processes in both normal and malignant cells. Previous studies have revealed mutations in the spliceosome complex, such as SF3B1, can cause increased AS frequencies in multiple myeloma (MM) patients, and patients with increased levels of AS are associated with a poor prognosis. Other frequently mutated genes involved in RNA processing include DIS3 and FAM46C, thus, systematically investigating other causes of AS abnormalities and pathologies in MM patients is highly necessary. Materials and Methods: RNA-seq data from 598 newly diagnosed MM patients from the MMRF CoMMpass study were utilized to generate AS comparisons. They were previously annotated for cytogenetic, copy number, and mutation data (version IA16). RNA-seq data were aligned to HG38 using STAR and Salmon. SUPPA2 was used for calling AS differences. For each identified AS event, the splicing level was defined by Percentage of Spliced-In (PSI) while the mean difference of splicing levels between two groups was measured by ΔPSI (dPSI) and by the P-value from independent T-tests against PSIs in the two groups. Filtering thresholds were determined to find high-quality differentially spliced events and were filtered for those also present in normal PCs (GSE110486). Geneset enrichment analysis was performed to identify dysregulated pathways caused by differential splicing and differential expression. Survival analysis was performed on clinical annotations of 598 NDMM patients while the Logrank test and Cox regression were used to evaluate the risk of AS and other genomic factors. Kaplan-Meier curves were plotted for various subgroups. Results: We compared 16 major cytogenetic subgroups, including Ig translocations (t(4;14), t(14;16), t(11;14)), hyperdiploidy, mutations in KRAS, NRAS, BRAF, FAM46C, SF3B1, DIS3 and TP53, combined events (t(4;14) plus DIS3 mutation), as well as those with biallelic abnormalities (DIS3, FAM46C, and TP53). Samples with SF3B1 hotspot mutations identified the greatest number of AS events (n=862), and samples with any SF3B1 mutation had approximately half as many. IGH translocations had an equivalent number of AS events to those with SF3B1 mutations, with t(14;16) having the most (n=587) followed by t(11;14) (n=366), and t(4;14) (n=256). We observed an increased number of significant AS events in bi-allelic DIS3 and FAM46C groups (n=404 and 171) compared to their mono-allelic abnormalities (n=114 and 35). As DIS3 mutations are enriched in the t(4;14) subgroup we also examined that interaction and found significantly more AS events (n=481; p<0.01) in the combination compared to either event alone. As expected, KRAS, NRAS and BRAF mutations did not have enrichment for AS events (n=2, 15, 23, respectively). The majority of AS events were unique to each subgroup, exemplifying the AS heterogeneity in these subgroups. Among overlapped events, an alternative first (AF) exon in ACACA was consistently more spliced in t(14;16), t(11;14) and t(4;14) groups (dPSI=0.18, 0.10, 0.12, P=2x10 -5, 2x10 -9, 5x10 -5). ACACA encodes an enzyme that significantly affects MM cell growth and viability, suggesting that similar regulations exist in the three translocation groups. Unique events were also detected including an AF event in MIB2 (E3 Ubiquitin Protein Ligase 2) in the t(11;14) group (dPSI=0.17, P=7x10 -14), and a skipped exon in UBXN4 (related to ER stress) in t(14;16) group (dPSI=0.1, P=3x10 -4). AS heterogeneity also leads to functional heterogeneity in the three groups. Besides commonly downregulated RNA catabolic processes, cell adhesion, migration and mobility related pathways are enriched pathways in t(14;16); cell growth related pathways in t(11;14); and ERK related pathways in t(4;14). High-risk events were identified through survival analysis and included a retained intron in RPS16 in the t(14;16) (Hazard Ratio (HR)=18.81, p=0.004). Similarly, high risk was associated with an AF event in DDX39B in t(11;14) (HR=2.62, p=0.001) and an AF event in COPA in t(4;14) (HR=6.29, p=0.001). Conclusion: AS is defined by multiple genomic events, including primary translocations and mutations in RNA processing genes, DIS3 and FAM46C, and interactions between genomic markers can increase AS. AS events contribute to outcome and some high risk AS events may serve as prognostic marker or potentially novel targets. Disclosures Walker: Sanofi: Speakers Bureau; Bristol Myers Squibb: Research Funding.

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High-throughput detection of RNA processing in bacteria

10.1101/073791 ◽

2016 ◽

Author(s):

Erin E Gill ◽

Luisa S Chan ◽

Geoffrey L Winsor ◽

Neil Dobson ◽

Raymond Lo ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

High Throughput ◽

Rna Processing ◽

Growth Conditions ◽

Rna Cleavage ◽

Sequence Motifs ◽

Rnase E ◽

Rna Seq ◽

Transcript Processing

Background: Understanding the RNA processing of an organism's transcriptome is an essential but challenging step in understanding its biology. Here we investigate with unprecedented detail the transcriptome of Pseudomonas aeruginosa PAO1, a medically important and innately multi-drug resistant bacterium. We systematically mapped RNA cleavage and dephosphorylation sites that result in 5'-monophosphate terminated RNA using a new high-throughput methodology called monophosphate RNA-Seq (pRNA-Seq). Transcriptional start sites (TSS) were also mapped using differential RNA-Seq (dRNA-Seq) and both datasets were compared to conventional RNA-Seq performed in a variety of growth conditions. Results: The pRNA-Seq transcript library revealed known tRNA, rRNA and tmRNA processing sites, together with previously uncharacterized RNA cleavage events that were found disproportionately near the 5' ends of transcripts associated with basic bacterial functions such as oxidative phosphorylation and purine metabolism. The majority (97%) of the processed mRNAs were cleaved at precise codon positions within defined sequence motifs indicative of distinct endonucleolytic activities. The most abundant of these motifs corresponded closely to an E. coli RNase E site previously established in vitro. Using the dRNA-Seq library, we performed an operon analysis and predicted 3,159 potential TSS. A correlation analysis uncovered 105 antiparallel pairs of TSS that were separated by 18 bp from each other and that were centered on a palindromic TAT(A/T)ATA motif, suggesting that such sites may provide a novel form of transcriptional regulation. TSS and RNA-Seq analysis allowed us to confirm expression of small non-coding RNAs (ncRNAs), many of which are differentially expressed in swarming and biofilm formation conditions. Conclusions: This study introduces pRNA-Seq methodology, which provides the first comprehensive, genome-wide survey of RNA processing in any organism. As a proof of concept, we have employed this technique to study the bacterium Pseudomonas aeruginosa and have discovered extensive transcript processing not previously appreciated. We have also gained novel insight into RNA maturation and turnover as well as a potential novel form of transcription regulation.

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Transcriptome-wide interrogation of the functional intronome by spliceosome profiling

10.1101/226894 ◽

2017 ◽

Author(s):

Weijun Chen ◽

Jill Moore ◽

Hakan Ozadam ◽

Hennady P. Shulha ◽

Nicholas Rhind ◽

...

Keyword(s):

Deep Sequencing ◽

Rna Processing ◽

Mrna Translation ◽

Ribosome Profiling ◽

Rna Seq ◽

Full Understanding ◽

Single Nucleotide ◽

Genome Wide ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

SUMMARYFull understanding of eukaryotic transcriptomes and how they respond to different conditions requires deep knowledge of all sites of intron excision. Although RNA-Seq provides much of this information, the low abundance of many spliced transcripts (often due to their rapid cytoplasmic decay) limits the ability of RNA-Seq alone to reveal the full repertoire of spliced species. Here we present “spliceosome profiling”, a strategy based on deep sequencing of RNAs co-purifying with late stage spliceosomes. Spliceosome profiling allows for unambiguous mapping of intron ends to single nucleotide resolution and branchpoint identification at unprecedented depths. Our data reveal hundreds of new introns in S. pombe and numerous others that were previously misannotated. By providing a means to directly interrogate sites of spliceosome assembly and catalysis genome-wide, spliceosome profiling promises to transform our understanding of RNA processing in the nucleus much like ribosome profiling has transformed our understanding mRNA translation in the cytoplasm.

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RNA-Seq analyses reveal CRISPR RNA processing and regulation patterns

Biochemical Society Transactions ◽

10.1042/bst20130129 ◽

2013 ◽

Vol 41 (6) ◽

pp. 1459-1463 ◽

Cited By ~ 11

Author(s):

Judith Zoephel ◽

Lennart Randau

Keyword(s):

Deep Sequencing ◽

Rna Processing ◽

Small Rnas ◽

Present Review ◽

Rna Seq ◽

Gradual Decline ◽

Crispr Array ◽

Rna Duplexes ◽

Rna Maturation ◽

Duplex Formation

In bacteria and archaea, RNA-Seq deep sequencing methodology allows for the detection of abundance and processing sites of the small RNAs that comprise a CRISPR (clustered regularly interspaced short palindromic repeats) RNome. Comparative analyses of these CRISPR RNome sets highlight conserved patterns that include the gradual decline of CRISPR RNA abundance from the leader-proximal to the leader-distal end. In the present review, we discuss exceptions to these patterns that indicate the extensive impact of individual spacer sequences on CRISPR array transcription and RNA maturation. Spacer sequences can contain promoter and terminator elements and can promote the formation of CRISPR RNA–anti-CRISPR RNA duplexes. In addition, potential RNA duplex formation with host tRNA was observed. These factors can influence the functionality of CRISPR–Cas (CRISPR-associated) systems and need to be considered in the design of synthetic CRISPR arrays.

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