Analysis Of RNA-seq Datasets Research Articles

Relevance of Thymic Stromal Lymphopoietin on the Pathogenesis of Glioblastoma: Role of the Neutrophil

Glioblastoma multiforme (GBM) is the most predominant and malignant primary brain tumor in adults. Thymic stromal lymphopoietin (TSLP), a cytokine primarily generated by activated epithelial cells, has recently garnered attention in cancer research. This study was aimed to elucidate the significance of TSLP in GBM cells and its interplay with the immune system, particularly focused on granulocyte neutrophils. Our results demonstrate that the tumor produces TSLP when stimulated with epidermal growth factor (EGF) in both the U251 cell line and the GBM biopsy (GBM-b). The relevance of the TSLP function was evaluated using a 3D spheroid model. Spheroids exhibited increased diameter, volume, and proliferation. In addition, TSLP promoted the generation of satellites surrounding the main spheroids and inhibited apoptosis in U251 treated with temozolomide (TMZ). Additionally, the co-culture of polymorphonuclear (PMN) cells from healthy donors with the U251 cell line in the presence of TSLP showed a reduction in apoptosis and an increase in IL-8 production. TSLP directly inhibited apoptosis in PMN from GBM patients (PMN-p). Interestingly, the vascular endothelial growth factor (VEGF) production was elevated in PMN-p compared with PMN from healthy donors. Under these conditions, TSLP also increased VEGF production, in PMN from healthy donors. Moreover, TSLP upregulated programed death-ligand 1 (PDL-1) expression in PMN cultured with U251. On the other hand, according to our results, the analysis of RNA-seq datasets from Illumina HiSeq 2000 sequencing platform performed with TIMER2.0 webserver demonstrated that the combination of TSLP with neutrophils decreases the survival of the patient. In conclusion, our results position TSLP as a possible new growth factor in GBM and indicate its modulation of the tumor microenvironment, particularly through its interaction with PMN.Graphical Protumoral activity of TSLP. Neutrophils (derived from GBM patients) and GBM cells (under EGF stimulus) not only produce TSLP but also express its receptor. TSLP induces PDL1 expression and decreases apoptosis on both GBM cells and neutrophils. TSLP also increases proliferation and satellite development on GBM cells, whereas favors more neutrophil infiltration by increasing IL8 production.

The RNA-binding proteins hnRNP H and F regulate splicing of a MYC-dependent HRAS exon in prostate cancer cells

The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.

Genome-wide comparative analysis of the valine glutamine motif containing genes in four Ipomoea species

BackgroundGenes with valine glutamine (VQ) motifs play an essential role in plant growth, development, and resistance to biotic and abiotic stresses. However, little information on the VQ genes in sweetpotato and other Ipomoea species is available.ResultsThis study identified 55, 58, 50 and 47 VQ genes from sweetpotato (I. batatas), I.triflida, I. triloba and I. nil, respectively. The phylogenetic analysis revealed that the VQ genes formed eight clades (I–VII), and the members in the same group exhibited similar exon–intron structure and conserved motifs distribution. The distribution of the VQ genes among the chromosomes of Ipomoea species was disproportional, with no VQ genes mapped on a few of each species' chromosomes. Duplication analysis suggested that segmental duplication significantly contributes to their expansion in sweetpotato, I.trifida, and I.triloba, while the segmental and tandem duplication contributions were comparable in I.nil. Cis-regulatory elements involved in stress responses, such as W-box, TGACG-motif, CGTCA-motif, ABRE, ARE, MBS, TCA-elements, LTR, and WUN-motif, were detected in the promoter regions of the VQ genes. A total of 30 orthologous groups were detected by syntenic analysis of the VQ genes. Based on the analysis of RNA-seq datasets, it was found that the VQ genes are expressed distinctly among different tissues and hormone or stress treatments. A total of 40 sweetpotato differentially expressed genes (DEGs) refer to biotic (sweetpotato stem nematodes and Ceratocystis fimbriata pathogen infection) or abiotic (cold, salt and drought) stress treatments were detected. Moreover, IbVQ8, IbVQ25 and IbVQ44 responded to the five stress treatments and were selected for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis, and the results were consistent with the transcriptome analysis.ConclusionsOur study may provide new insights into the evolution of VQ genes in the four Ipomoea genomes and contribute to the future molecular breeding of sweetpotatoes.

Confounding factors in profiling of locus-specific human endogenous retrovirus (HERV) transcript signatures in primary T cells using multi-study-derived datasets

BackgroundHuman endogenous retroviruses (HERV) are repetitive sequence elements and a substantial part of the human genome. Their role in development has been well documented and there is now mounting evidence that dysregulated HERV expression also contributes to various human diseases. While research on HERV elements has in the past been hampered by their high sequence similarity, advanced sequencing technology and analytical tools have empowered the field. For the first time, we are now able to undertake locus-specific HERV analysis, deciphering expression patterns, regulatory networks and biological functions of these elements. To do so, we inevitable rely on omics datasets available through the public domain. However, technical parameters inevitably differ, making inter-study analysis challenging. We here address the issue of confounding factors for profiling locus-specific HERV transcriptomes using datasets from multiple sources.MethodsWe collected RNAseq datasets of CD4 and CD8 primary T cells and extracted HERV expression profiles for 3220 elements, resembling most intact, near full-length proviruses. Looking at sequencing parameters and batch effects, we compared HERV signatures across datasets and determined permissive features for HERV expression analysis from multiple-source data.ResultsWe could demonstrate that considering sequencing parameters, sequencing-depth is most influential on HERV signature outcome. Sequencing samples deeper broadens the spectrum of expressed HERV elements. Sequencing mode and read length are secondary parameters. Nevertheless, we find that HERV signatures from smaller RNAseq datasets do reliably reveal most abundantly expressed HERV elements. Overall, HERV signatures between samples and studies overlap substantially, indicating a robust HERV transcript signature in CD4 and CD8 T cells. Moreover, we find that measures of batch effect reduction are critical to uncover genic and HERV expression differences between cell types. After doing so, differences in the HERV transcriptome between ontologically closely related CD4 and CD8 T cells became apparent.ConclusionIn our systematic approach to determine sequencing and analysis parameters for detection of locus-specific HERV expression, we provide evidence that analysis of RNAseq datasets from multiple studies can aid confidence of biological findings. When generating de novo HERV expression datasets we recommend increased sequence depth ( > = 100 mio reads) compared to standard genic transcriptome pipelines. Finally, batch effect reduction measures need to be implemented to allow for differential expression analysis.

Tip60's Novel RNA-Binding Function Modulates Alternative Splicing of Pre-mRNA Targets Implicated in Alzheimer's Disease.

The severity of Alzheimer's disease (AD) progression involves a complex interplay of genetics, age, and environmental factors orchestrated by histone acetyltransferase (HAT)-mediated neuroepigenetic mechanisms. While disruption of Tip60 HAT action in neural gene control is implicated in AD, alternative mechanisms underlying Tip60 function remain unexplored. Here, we report a novel RNA binding function for Tip60 in addition to its HAT function. We show that Tip60 preferentially interacts with pre-mRNAs emanating from its chromatin neural gene targets in the Drosophila brain and this RNA binding function is conserved in human hippocampus and disrupted in Drosophila brains that model AD pathology and in AD patient hippocampus of either sex. Since RNA splicing occurs co-transcriptionally and alternative splicing (AS) defects are implicated in AD, we investigated whether Tip60-RNA targeting modulates splicing decisions and whether this function is altered in AD. Replicate multivariate analysis of transcript splicing (rMATS) analysis of RNA-Seq datasets from wild-type and AD fly brains revealed a multitude of mammalian-like AS defects. Strikingly, over half of these altered RNAs are identified as bona-fide Tip60-RNA targets that are enriched for in the AD-gene curated database, with some of these AS alterations prevented against by increasing Tip60 in the fly brain. Further, human orthologs of several Tip60-modulated splicing genes in Drosophila are well characterized aberrantly spliced genes in human AD brains, implicating disruption of Tip60's splicing function in AD pathogenesis. Our results support a novel RNA interaction and splicing regulatory function for Tip60 that may underly AS impairments that hallmark AD etiology.SIGNIFICANCE STATEMENT Alzheimer's disease (AD) has recently emerged as a hotbed for RNA alternative splicing (AS) defects that alter protein function in the brain yet causes remain unclear. Although recent findings suggest convergence of epigenetics with co-transcriptional AS, whether epigenetic dysregulation in AD pathology underlies AS defects remains unknown. Here, we identify a novel RNA interaction and splicing regulatory function for Tip60 histone acetyltransferase (HAT) that is disrupted in Drosophila brains modeling AD pathology and in human AD hippocampus. Importantly, mammalian orthologs of several Tip60-modulated splicing genes in Drosophila are well characterized aberrantly spliced genes in human AD brain. We propose that Tip60-mediated AS modulation is a conserved critical posttranscriptional step that may underlie AS defects now characterized as hallmarks of AD.

Operation of the Atypical Canonical Bone Morphogenetic Protein Signaling Pathway During Early Human Odontogenesis.

Bone morphogenetic protein (BMP) signaling plays essential roles in the regulation of early tooth development. It is well acknowledged that extracellular BMP ligands bind to the type I and type II transmembrane serine/threonine kinase receptor complexes to trigger the BMP signaling pathway. Then, the receptor-activated Smad1/5/8 in cytoplasm binds to Smad4, the central mediator of the canonical BMP signaling pathway, to form transfer complexes for entering the nucleus and regulating target gene expression. However, a recent study revealed the functional operation of a novel BMP-mediated signaling pathway named the atypical BMP canonical signaling pathway in mouse developing tooth, which is Smad1/5/8 dependent but Smad4 independent. In this study, we investigated whether this atypical BMP canonical signaling is conserved in human odontogenesis. We showed that pSMAD1/5/8 is required for the expression of Msh homeobox 1 (MSX1), a well-defined BMP signaling target gene, in human dental mesenchyme, but the typical BMP canonical signaling is in fact not operating in the early human developing tooth, as evidenced by the absence of pSMAD1/5/8-SMAD4 complexes in the dental mesenchyme and translocation of pSMAD1/5/8, and the expression of MSX1 induced by BMP4 is mothers against decapentaplegic homolog 4 (SMAD4)-independent in human dental mesenchymal cells. Moreover, integrative analysis of RNA-Seq data sets comparing the transcriptome profiles of human dental mesenchymal cells with and without SMAD4 knockdown by siRNA displays unchanged expression profiles of pSMAD1/5/8 downstream target genes, further affirming the functional operation of the atypical canonical BMP signaling pathway in a SMAD1/5/8-dependent but SMAD4-independent manner in the dental mesenchyme during early odontogenesis in humans.

Identification of wheat DNA-binding with one finger (Dof) transcription factor in genomic sequence of Triticum aestivum chromosome 2

The DNA- binding with one finger (Dof) transcription factors plays an important role in various biological functions in plants such as photosynthetic carbon assimilation, stress response, and flowering time. The information regarding the Dof protein is limited in wheat due to lack of wheat genomic data. In this regard, we conducted TaDof gene identification in chromosome 2 and identified eighteen TaDof proteins. These putative genes were mapped on the 2A, 2B and 2D chromosome. The motif analysis confirms the presence of the DNA-binding domain containing C2C2-type zinc figure motif at N-terminal and a transcriptional regulation domain at the C-terminal in the identified sequence. We compared TaDof in rice, wheat, maize, sorghum and performed phylogenetic analysis. These genes were classified into five groups based on phylogenetic analysis supported by high bootstrap values. Tissue- and developmental stage -specific expression analysis of RNA-seq datasets revealed the constitutive as well as the inductive response of TaDof genes in different tissues of wheat. RT-PCR analysis of five selected TaDof TFs were confirmed their responsiveness to drought, heat and salt stress. This study provides valuable information for understanding not only the potential function of Dof genes in regulating abiotic stresses, but also will be basis for exploring the function and evolution studies of more candidate TaDof genes.

Transcriptomic analysis of the heat-stress response in Boechera depauperata and Arabidopsis reveals a distinct and unusual heat-stress response in Boechera

Global surface temperatures are expected to rise throughout the 21st century, and negatively impact plant growth and reproduction. Thus, it is imperative that we deepen our understanding of plant thermotolerance. The examination of native plant species that have evolved tolerance to high temperatures can provide crucial information on how plants can adapt to climate change. Boechera (Brassicaceae), a large genus that is native to North America, is highly thermotolerant, and can maintain photosynthetic activity at high temperatures. Here we report results of transcriptomic studies that seek to reveal possible thermotolerance mechanisms in B. depauperata (A.Nelson & P.B.Kenn.) Windham & Al-Shehbaz. Analysis of RNA-seq datasets from heat stressed B. depauperata and Arabidopsis thaliana (L.) Heynh. plants identified significant differences in how each of these species responds to identical heat stress conditions. The most highly upregulated heat-stress genes in A. thaliana includes the well-characterized heat-shock genes. In contrast, the Boechera heat-stress response is composed of: novel genes that lack orthologs in other genomes; genes coding for proteins of uncharacterized function; and genes coding for proteins associated with the unfolded protein and endoplasmic reticulum stress responses. In addition, genes that are protective of photosynthetic capacity are also differentially upregulated in B. depauperata.

Expression profiling of WD40 family genes including DDB1- and CUL4- associated factor (DCAF) genes in mice and human suggests important regulatory roles in testicular development and spermatogenesis

BackgroundThe WD40-repeat containing proteins, including DDB1–CUL4-associated factors (DCAFs), are abundant and conserved proteins that play important roles in different cellular processes including spermatogenesis. DCAFs are subset of WD40 family proteins that contain WDxR motif and have been proposed to function as substrate receptor for Cullin4-RING-based E3 ubiquitin ligase complexes to recruit diverse proteins for ubiquitination, a vital process in spermatogenesis. Large number of WD40 genes has been identified in different species including mouse and human. However, a systematic expression profiling of WD40 genes in different tissues of mouse and human has not been investigated. We hypothesize that large number of WD40 genes may express highly or specifically in the testis, where their expression is uniquely regulated during testis development and spermatogenesis. Therefore, the objective of this study is to mine and characterize expression patterns of WD40 genes in different tissues of mouse and human with particular emphasis on DCAF genes expressions during mouse testicular development.ResultsPublically available RNA sequencing (RNA seq) data mining identified 347 and 349 WD40 genes in mouse and human, respectively. Hierarchical clustering and heat map analyses of RNA seq datasets revealed differential expression patterns of WD40 genes with around 60–73% of the genes were highly or specifically expressed in testis. Similarly, around 74–83% of DCAF genes were predominantly or specifically expressed in testis. Moreover, WD40 genes showed distinct expression patterns during embryonic and postnatal testis development in mice. Finally, different germ cell populations of testis showed specific patterns of WD40 genes expression. Predicted gene ontology analyses revealed more than 80% of these proteins are implicated in cellular, metabolic, biological regulation and cell localization processes.ConclusionsWe have identified large number of WD40 family genes that are highly or specifically expressed in the testes of mouse and human. Moreover, WD40 genes have distinct expression patterns during embryonic and postnatal development of the testis in mice. Further, different germ cell populations within the testis showed specific patterns of WD40 genes expression. These results provide foundation for further research towards understanding the functional genomics and molecular mechanisms of mammalian testis development and spermatogenesis.

Large-scale discovery of male reproductive tract-specific genes through analysis of RNA-seq datasets

BackgroundThe development of a safe, effective, reversible, non-hormonal contraceptive method for men has been an ongoing effort for the past few decades. However, despite significant progress on elucidating the function of key proteins involved in reproduction, understanding male reproductive physiology is limited by incomplete information on the genes expressed in reproductive tissues, and no contraceptive targets have so far reached clinical trials. To advance product development, further identification of novel reproductive tract-specific genes leading to potentially druggable protein targets is imperative.ResultsIn this study, we expand on previous single tissue, single species studies by integrating analysis of publicly available human and mouse RNA-seq datasets whose initial published purpose was not focused on identifying male reproductive tract-specific targets. We also incorporate analysis of additional newly acquired human and mouse testis and epididymis samples to increase the number of targets identified. We detected a combined total of 1178 genes for which no previous evidence of male reproductive tract-specific expression was annotated, many of which are potentially druggable targets. Through RT-PCR, we confirmed the reproductive tract-specific expression of 51 novel orthologous human and mouse genes without a reported mouse model. Of these, we ablated four epididymis-specific genes (Spint3, Spint4, Spint5, and Ces5a) and two testis-specific genes (Pp2d1 and Saxo1) in individual or double knockout mice generated through the CRISPR/Cas9 system. Our results validate a functional requirement for Spint4/5 and Ces5a in male mouse fertility, while demonstrating that Spint3, Pp2d1, and Saxo1 are each individually dispensable for male mouse fertility.ConclusionsOur work provides a plethora of novel testis- and epididymis-specific genes and elucidates the functional requirement of several of these genes, which is essential towards understanding the etiology of male infertility and the development of male contraceptives.

COVID-19 Hyperinflammation: What about Neutrophils?

COVID-19 is often related to hyperinflammation that drives lung or multiorgan injury. The immunopathological mechanisms that cause excessive inflammation are under investigation and constantly updated. Here, a gene network approach was used on recently published data sets to identify possible COVID-19 inflammatory mechanisms and bioactive genes. First, network analysis of putative SARS-CoV-2 cellular receptors led to the mining of a neutrophil-response signature and relevant inflammatory genes. Second, analysis of RNA-seq data sets of lung cells infected with SARS-CoV-2 revealed that infected cells expressed neutrophil-attracting chemokines. Third, analysis of RNA-seq data sets of bronchoalveolar lavage fluid cells from COVID-19 patients identified upregulation of neutrophil genes and chemokines. Different inflammatory genes mined here, including TNFR, IL-8, CXCR1, CXCR2, ADAM10, GPR84, MME, ANPEP, and LAP3, might be druggable targets in efforts to limit SARS-CoV-2 inflammation in severe clinical cases. The possible role of neutrophils in COVID-19 inflammation needs to be studied further.

On the utility of RNA sample pooling to optimize cost and statistical power in RNA sequencing experiments

BackgroundIn gene expression studies, RNA sample pooling is sometimes considered because of budget constraints or lack of sufficient input material. Using microarray technology, RNA sample pooling strategies have been reported to optimize both the cost of data generation as well as the statistical power for differential gene expression (DGE) analysis. For RNA sequencing, with its different quantitative output in terms of counts and tunable dynamic range, the adequacy and empirical validation of RNA sample pooling strategies have not yet been evaluated. In this study, we comprehensively assessed the utility of pooling strategies in RNA-seq experiments using empirical and simulated RNA-seq datasets.ResultThe data generating model in pooled experiments is defined mathematically to evaluate the mean and variability of gene expression estimates. The model is further used to examine the trade-off between the statistical power of testing for DGE and the data generating costs. Empirical assessment of pooling strategies is done through analysis of RNA-seq datasets under various pooling and non-pooling experimental settings. Simulation study is also used to rank experimental scenarios with respect to the rate of false and true discoveries in DGE analysis. The results demonstrate that pooling strategies in RNA-seq studies can be both cost-effective and powerful when the number of pools, pool size and sequencing depth are optimally defined.ConclusionFor high within-group gene expression variability, small RNA sample pools are effective to reduce the variability and compensate for the loss of the number of replicates. Unlike the typical cost-saving strategies, such as reducing sequencing depth or number of RNA samples (replicates), an adequate pooling strategy is effective in maintaining the power of testing DGE for genes with low to medium abundance levels, along with a substantial reduction of the total cost of the experiment. In general, pooling RNA samples or pooling RNA samples in conjunction with moderate reduction of the sequencing depth can be good options to optimize the cost and maintain the power.

Transcriptomic Response of In Vitro Potato (Solanum tuberosum L.) to Piezoelectric Ultrasound

As part of a wider project to assess the impact of ultrasound on in vitro plant growth, this paper aimed to determine whether the application of piezoelectric ultrasound (PE-US) would induce changes to the transcriptome of in vitro potato (Solanum tuberosum L.). After exposing explants (single-node segments with a single leaf) to PE-US (35 kHz; 70 W) for 20 min, the effect of this stressor was determined at 0 h, 24 h, 48 h, 1 week, and 4 weeks to assess the possible immediate and residual effects of PE-US on the potato transcriptome. After mRNA was isolated, bioinformatic processing and analysis of RNA-seq datasets, KEGG analysis revealed that 24% of up-regulated biological processes were a response to stress immediately after the application of PE-US (reducing to 11% and 9% at 48 h and 4 weeks, respectively). After assessing a total of 40,430 genes for expression intensity at these five time intervals, it was found that 138, 72, 18, 5, and 59 differentially expressed genes (DEGs), respectively, were significantly up-regulated, while 6, 82, 96, 172, and 107 DEGs, respectively, were down-regulated. DEGs coding for universal stress protein, chitinase, catalase, zinc finger proteins, 21 transcription factors, glutathione S-transferase, and 17 heat shock proteins, mainly Hsp70, Hsp20, and Hsp90 proteins, and possibly the first of such discovery in US-based plant stress research, was detected. Following the validation of RNA-seq data by RT-qPCR, between SeqMonk LFC and RT-qPCR LFC, the Spearman and Pearson correlation coefficients were 0.86 and 0.90, respectively. Plantlets exposed to PE-US had significantly shorter shoots but significantly longer roots as well as higher shoot and root fresh weight, while chl b and total chl were significantly lower but the chl a/b ratio was significantly higher in plantlets exposed to PE-US. PE-US thus constituted an acute abiotic stress, but by 4 weeks, in vitro potato plantlets managed to mitigate the stress through the production of antioxidant enzyme systems and other functional metabolic changes.

Detection of Differentially Expressed Cleavage Site Intervals Within 3' Untranslated Regions Using CSI-UTR Reveals Regulated Interaction Motifs.

The length of untranslated regions at the 3′ end of transcripts (3′UTRs) is regulated by alternate polyadenylation (APA). 3′UTRs contain regions that harbor binding motifs for regulatory molecules. However, the mechanisms that coordinate the 3′UTR length of specific groups of transcripts are not well-understood. We therefore developed a method, CSI-UTR, that models 3′UTR structure as tandem segments between functional alternative-polyadenylation sites (termed cleavage site intervals—CSIs). This approach facilitated (1) profiling of 3′UTR isoform expression changes and (2) statistical enrichment of putative regulatory motifs. CSI-UTR analysis is UTR-annotation independent and can interrogate legacy data generated from standard RNA-Seq libraries. CSI-UTR identified a set of CSIs in human and rodent transcriptomes. Analysis of RNA-Seq datasets from neural tissue identified differential expression events within 3′UTRs not detected by standard gene-based differential expression analyses. Further, in many instances 3′UTR and CDS from the same gene were regulated differently. This modulation of motifs for RNA-interacting molecules with potential condition-dependent and tissue-specific RNA binding partners near the polyA signal and CSI junction may play a mechanistic role in the specificity of alternative polyadenylation.Source code, CSI BED files and example datasets are available at: https://github.com/UofLBioinformatics/CSI-UTR

The Development and Use of Scalable Systems for Studying Aberrant Splicing in SF3B1-Mutant CLL.

Mutational landscape of CLL is now known to include recurrent non-synonymous mutations in SF3B1, a core splicing factor. About 5-10% of newly diagnosed CLL harbor these mutations which are typically limited to HEAT domains in the carboxyl-terminus of the protein. Importantly, the mutations are not specific to CLL but also present in several unrelated clonal disorders. Analysis of patient samples and cell lines has shown the primary splicing aberration in SF3B1-mutant cells to the use of novel or "cryptic" 3' splice sites (3SS). Advances in genome-editing and next-generation sequencing (NGS) have allowed development of isogenic models and detailed analysis of changes to the transcriptome with relative ease. In this manuscript, we focus on two relevant methods to study splicing factor mutations in CLL: development of isogenic scalable cell lines and informatics analysis of RNA-Seq datasets.

Analysis of RNA-Seq datasets reveals enrichment of tissue-specific splice variants for nuclear envelope proteins

ABSTRACTLaminopathies yield tissue-specific pathologies, yet arise from mutation of ubiquitously-expressed genes. A little investigated hypothesis to explain this is that the mutated proteins or their partners have tissue-specific splice variants. To test this, we analyzed RNA-Seq datasets, finding novel isoforms or isoform tissue-specificity for: Lap2, linked to cardiomyopathy; Nesprin 2, linked to Emery-Dreifuss muscular dystrophy and Lmo7, that regulates the Emery-Dreifuss muscular dystrophy linked emerin gene. Interestingly, the muscle-specific Lmo7 exon is rich in serine phosphorylation motifs, suggesting regulatory function. Muscle-specific splice variants in non-nuclear envelope proteins linked to other muscular dystrophies were also found. Nucleoporins tissue-specific variants were found for Nup54, Nup133, Nup153 and Nup358/RanBP2. RT-PCR confirmed novel Lmo7 and RanBP2 variants and specific knockdown of the Lmo7 variantreduced myogenic index. Nuclear envelope proteins were enriched for tissue-specific splice variants compared to the rest of the genome, suggesting that splice variants contribute to its tissue-specific functions.

RNA Sequencing (RNA-Seq) Reveals Extremely Low Levels of Reticulocyte-Derived Globin Gene Transcripts in Peripheral Blood From Horses (Equus caballus) and Cattle (Bos taurus).

RNA-seq has emerged as an important technology for measuring gene expression in peripheral blood samples collected from humans and other vertebrate species. In particular, transcriptomics analyses of whole blood can be used to study immunobiology and develop novel biomarkers of infectious disease. However, an obstacle to these methods in many mammalian species is the presence of reticulocyte-derived globin mRNAs in large quantities, which can complicate RNA-seq library sequencing and impede detection of other mRNA transcripts. A range of supplementary procedures for targeted depletion of globin transcripts have, therefore, been developed to alleviate this problem. Here, we use comparative analyses of RNA-seq data sets generated from human, porcine, equine, and bovine peripheral blood to systematically assess the impact of globin mRNA on routine transcriptome profiling of whole blood in cattle and horses. The results of these analyses demonstrate that total RNA isolated from equine and bovine peripheral blood contains very low levels of globin mRNA transcripts, thereby negating the need for globin depletion and greatly simplifying blood-based transcriptomic studies in these two domestic species.

BioSpotlight / Citations

BioSpotlight / Citations

Abstract B43: Global upregulation of ribosomal protein gene pathway in normal cerebellar progenitor development and in pathogenesis of sonic hedgehog subtype medulloblastomas

Abstract Co-regulated transcription of ribosomal protein genes (RPGs) is at the heart of efficient ribosome biogenesis; however, mechanisms that regulate RPG pathways in relation to normal vertebrate development and disease are not well understood. Granule progenitors in the external granule layer (EGL) of the developing mouse cerebellum show a sharp proliferative spike during the first two weeks after birth in a sonic hedgehog (Shh)-dependent manner. Medulloblastomas (MBs) constitute the most common malignant pediatric brain tumor originating in the cerebellum. In particular, Shh subtype MBs result from unregulated proliferation of granule progenitors, and understanding mechanisms underlying their proliferative capacity can provide insights into MB pathogenesis and therapeutics. Interestingly, increased ribosomal protein (RP) levels in MBs have been associated with poor patient survival. Here, we describe an unexpected role of global upregulation of the RPG pathway in normal cerebellar granule progenitor development and in pathogenesis of Shh subtype MBs. Using unbiased RNA-seq analysis, we detected global transcriptional upregulation of RPG pathway in mouse cerebellum during postnatal development compared to adults. In situ hybridization and immunohistochemistry of individual RPs demonstrated increased transcript and protein levels of individual RPs specifically in the EGL, respectively. Increased RP levels in EGL progenitors resulted predominantly by transcriptional mechanisms, as opposed to post-mitotic purkinje neurons showing selective upregulation of RPG protein but not transcript levels. Translational analysis of nascent polypeptide chains by incorporation of O-propargyl-puromycin by flow cytometry and immunofluorescence revealed increased protein translation in EGL with respect to rest of the neonatal cerebellum, suggesting functional ribosomal biogenesis in addition to global upregulation of RPG pathway. To examine if reducing RP levels affect normal tissue homeostasis, we analyzed brain and cerebellum development in a hypomorphic allele of an individual RP (Rpl24 Bst/+). Although RPG pathway was transcriptionally downegulated in Rpl24 Bst/+ neonatal cerebellum with respect to wild type, gross cerebellar morphology and granule neuron numbers in adult animals were not affected. In addition, neurogenesis in the subventricular zone of the lateral ventricle and subgranular zone of the dentate gyrus of Rpl24 Bst/+ mice were not affected. Thus, partial reduction of RPs does not impact upon normal brain development. Interestingly, Shh subtype MBs in mice demonstrated global upregulation of RPG transcripts and increased individual protein levels in comparison to postnatal cerebellum, which already has higher levels of RPG pathway compared to adults. Human Shh-MB samples also demonstrated higher RP levels with respect to normal cerebellar tissue. Analysis of RNA-seq datasets in TCGA suggested that global transcriptional upregulation of the RPG pathway is not a common feature of all human tumors, in particular only ~50% of tumor subtypes showed global upregulation. However, global upregulation of RPG pathway in Shh-MBs is remarkably significant both in the extent and severity of the increase. Interestingly, we observed reduced tumor size in mice with Rpl24 hypomorphism compared with control mice expressing only constitutively active Smoothened (SmoM2) in neural stem/progenitors, suggesting that the RPG pathway determines tumorigenic burden in Shh-MBs. Thus, unlike cerebellar progenitor development, Shh-MBs show dependency on global RPG transcription. Characterizing the RPG pathway in human MB subtypes with respect to patient prognosis and tumor recurrence, and/or therapeutic strategies targeting ribosomal biogenesis might be transformative in developing effective Shh-MB therapies. Citation Format: Issei Shimada, Somatilaka Bandarigoda, John Shelton, Zhenyu Xuan, Veena Rajaram, Saikat Mukhopadhyay. Global upregulation of ribosomal protein gene pathway in normal cerebellar progenitor development and in pathogenesis of sonic hedgehog subtype medulloblastomas. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B43.

A computational method for estimating the PCR duplication rate in DNA and RNA-seq experiments

BackgroundPCR amplification is an important step in the preparation of DNA sequencing libraries prior to high-throughput sequencing. PCR amplification introduces redundant reads in the sequence data and estimating the PCR duplication rate is important to assess the frequency of such reads. Existing computational methods do not distinguish PCR duplicates from “natural” read duplicates that represent independent DNA fragments and therefore, over-estimate the PCR duplication rate for DNA-seq and RNA-seq experiments.ResultsIn this paper, we present a computational method to estimate the average PCR duplication rate of high-throughput sequence datasets that accounts for natural read duplicates by leveraging heterozygous variants in an individual genome. Analysis of simulated data and exome sequence data from the 1000 Genomes project demonstrated that our method can accurately estimate the PCR duplication rate on paired-end as well as single-end read datasets which contain a high proportion of natural read duplicates. Further, analysis of exome datasets prepared using the Nextera library preparation method indicated that 45–50% of read duplicates correspond to natural read duplicates likely due to fragmentation bias. Finally, analysis of RNA-seq datasets from individuals in the 1000 Genomes project demonstrated that 70–95% of read duplicates observed in such datasets correspond to natural duplicates sampled from genes with high expression and identified outlier samples with a 2-fold greater PCR duplication rate than other samples.ConclusionsThe method described here is a useful tool for estimating the PCR duplication rate of high-throughput sequence datasets and for assessing the fraction of read duplicates that correspond to natural read duplicates. An implementation of the method is available at https://github.com/vibansal/PCRduplicates.