Identification of novel transcripts and peptides in developing murine lens.
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ABSTRACT:
SUBMITTER: Khan SY
PROVIDER: S-EPMC6057992 | biostudies-literature | 2018 Jul
REPOSITORIES: biostudies-literature
Publications
Identification of novel transcripts and peptides in developing murine lens.
Khan Shahid Y SY Ali Muhammad M Kabir Firoz F Chen Ruiqiang R Na Chan Hyun CH Lee Mei-Chong W MW Pourmand Nader N Hackett Sean F SF Riazuddin S Amer SA
Scientific reports 20180724 1
We previously investigated the transcriptome and proteome profiles of the murine ocular lens at six developmental time points including two embryonic (E15 and E18) and four postnatal time points (P0, P3, P6, and P9). Here, we extend our analyses to identify novel transcripts and peptides in developing mouse lens. We identified a total of 9,707 novel transcripts and 325 novel fusion genes in developing mouse lens. Additionally, we identified 13,281 novel alternative splicing (AS) events in mouse ...[more]
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Project description:Lens development involves a complex and highly orchestrated regulatory program. Here, we investigate the transcriptomic alterations and splicing events during mouse lens formation using RNA-seq data from multiple developmental stages, and construct a molecular portrait of known and novel transcripts. We show that the extent of novelty of expressed transcripts decreases significantly in post-natal lens compared to embryonic stages. Characterization of novel transcripts into partially novel transcripts (PNTs) and completely novel transcripts (CNTs) (novelty score???70%) revealed that the PNTs are both highly conserved across vertebrates and highly expressed across multiple stages. Functional analysis of PNTs revealed their widespread role in lens developmental processes while hundreds of CNTs were found to be widely expressed and predicted to encode for proteins. We verified the expression of four CNTs across stages. Examination of splice isoforms revealed skipped exon and retained intron to be the most abundant alternative splicing events during lens development. We validated by RT-PCR and Sanger sequencing, the predicted splice isoforms of several genes Banf1, Cdk4, Cryaa, Eif4g2, Pax6, and Rbm5. Finally, we present a splicing browser Eye Splicer ( http://www.iupui.edu/~sysbio/eye-splicer/ ), to facilitate exploration of developmentally altered splicing events and to improve understanding of post-transcriptional regulatory networks during mouse lens development.
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Project description:Non-coding RNAs (ncRNAs) are emerging as an important player in the regulation of genome integrity and gene expression, and they have been implicated in the pathogenesis of many diseases. The aim of the present study is to identify the repertoire of ncRNAs expressed in the developing mouse lens. We previously reported the mouse lens transcriptome, including mRNA and microRNA (miRNA) profiling at two embryonic (E15 and E18) and four postnatal (P0, P3, P6, and P9) time points. We analyzed the data from small RNA-Seq and mRNA-Seq libraries to investigate the ncRNA profile. Our analysis revealed expression of 12 different classes of ncRNA in the murine lens at six developmental time points. Annotation of small RNA data showed expression of 1,756 antisense ncRNA (asncRNA) in the mouse lens transcriptome. Likewise, we identified 82 P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA), 345 transfer RNA (tRNA), 12 small nuclear RNA (snRNA), 167 small nucleolar RNA (snoRNA), 19 small Cajal body-specific RNA (scaRNA), six ribosomal RNA (rRNA), 18 tRNA-like structures, one MALAT1-associated small cytoplasmic RNA (mascRNA), one Vault RNA (vtRNA), and one Y RNA expressed in the developing mouse lens. In parallel, bioinformatic investigation of mRNA-Seq data identified expression of 1,952 long intergenic ncRNA (lincRNA) in the developing mouse lens. In conclusion, we report a comprehensive ncRNA profile in the murine lens at six developmental time points. To the best of our knowledge, this is first report investigating different classes of ncRNAs in the developing mouse lens and will be monumental in elucidating processes essential for the development of the ocular lens and the maintenance of its transparency.
| S-EPMC5295360 | biostudies-literature
Project description:PurposeWe previously completed a comprehensive profile of the mouse lens transcriptome. Here, we investigate the proteome of the mouse lens through mass spectrometry-based protein sequencing at the same embryonic and postnatal time points.MethodsWe extracted mouse lenses at embryonic day 15 (E15) and 18 (E18) and postnatal day 0 (P0), 3 (P3), 6 (P6), and 9 (P9). The lenses from each time point were preserved in three distinct pools to serve as biological replicates for each developmental stage. The total cellular protein was extracted from the lens, digested with trypsin, and labeled with isobaric tandem mass tags (TMT) for three independent TMT experiments.ResultsA total of 5404 proteins were identified in the mouse ocular lens in at least one TMT set, 4244 in two, and 3155 were present in all three TMT sets. The majority of the proteins exhibited steady expression at all six developmental time points; nevertheless, we identified 39 proteins that exhibited an 8-fold differential (higher or lower) expression during the developmental time course compared to their respective levels at E15. The lens proteome is composed of diverse proteins that have distinct biological properties and functional characteristics, including proteins associated with cataractogenesis and autophagy.ConclusionsWe have established a comprehensive profile of the developing murine lens proteome. This repository will be helpful in identifying critical components of lens development and processes essential for the maintenance of its transparency.
| S-EPMC5769801 | biostudies-literature
Project description:PurposeTranscriptome is the entire repertoire of transcripts present in a cell at any particular time. We undertook a next-generation whole transcriptome sequencing approach to gain insight into the transcriptional landscape of the developing mouse lens.MethodsWe ascertained mouse lenses at six developmental time points including two embryonic (E15 and E18) and four postnatal stages (P0, P3, P6, and P9). The ocular tissue at each time point was maintained as two distinct pools serving as biological replicates for each developmental stage. The mRNA and small RNA libraries were paired-end sequenced on Illumina HiSeq 2000 and subsequently analyzed using bioinformatics tools.ResultsMapping of mRNA and small RNA libraries generated 187.56 and 154.22 million paired-end reads, respectively. We detected a total of 14,465 genes in the mouse ocular lens at the above-mentioned six developmental stages. Of these, 46 genes exhibited a 40-fold differential (higher or lower) expression at one the five developmental stages (E18, P0, P3, P6, and P9) compared with their expression level at E15. Likewise, small RNA profiling identified 379 microRNAs (miRNAs) expressed in mouse lens at six developmental time points. Of these, 49 miRNAs manifested an 8-fold differential (higher or lower) expression at one the five developmental stages, as mentioned above compared with their expression level at E15.ConclusionsWe report a comprehensive profile of developing murine lens transcriptome including both mRNA and miRNA through next-generation RNA sequencing. A complete repository of the lens transcriptome of six developmental time points will be monumental in elucidating processes essential for the development of the ocular lens and maintenance of its transparency.
| S-EPMC4525677 | biostudies-literature
Project description:The proteome is a term used for the entire catalog of proteins present in a cell at any time. In here, we investigated the proteome of developing mouse lens through mass spectrometry-based protein sequencing. We extracted mouse lenses at six developmental time points, which included two embryonic (E15 and E18) and four postnatal stages (P0, P3, P6, and P9). The lenses from each time point were preserved in three distinct pools to serve as biological replicates for each developmental stage. The total cellular protein was extracted from the lens, digested with trypsin and labeled with 6-plex isobaric tandem mass tags (TMT) for three independent 6-plex TMT experiments. A total of 6,117 proteins were identified in the mouse ocular lens in at least one of the above-mentioned six developmental time points. Of these, 6,117 proteins were present in one TMT set, 4,325 in two, and 2,972 were present in three TMT sets, respectively. Majority of the proteins exhibit steady expression; however, we identified 162 proteins that exhibited an 8-fold differential (higher or lower) expression during the developmental time course compared to their levels at E15. The lens proteome is comprised of diverse proteins that have distinct biological properties and functional characteristics including a total of 404 proteins that have been associated with autophagy. We have established a comprehensive profile of the developing murine lens proteome. This repository will be helpful in identifying critical components of lens development and processes essential for the maintenance of its transparency.
2018-01-22 | PXD006381 | Pride
Project description:Purpose: Transcriptome is the entire repertoire of all transcripts present in a cell at any particular time. We undertook next-generation whole transcriptome sequencing approach to gain insight of the transcriptional landscape of the developing mouse lens. Methods: We ascertained mice lenses at six developmental time points including two embryonic (E15 and E18) and four postnatal stages (P0, P3, P6, and P9). The ocular tissue at each time point was maintained as two distinct pools serving as biological replicates for each developmental stage. The mRNA and small RNA libraries were paired-end sequenced on Illumina HiSeq 2000 and subsequently analyzed using bioinformatics tools. Results: Mapping of mRNA and small RNA libraries generated 187.56 and 154.22 million paired-end reads, respectively. We detected a total of 14,465 genes in the mouse ocular lens. Of these, 46 genes exhibited 40-fold differential expression compared to transcriptional levels at E15. Likewise, small RNA profiling identified 379 microRNAs (miRNAs) expressed in mouse lens. Of these, 49 miRNAs manifested an 8-fold or higher differential expression when compared, as above to the microRNA expression at E15. Conclusion: We report the first comprehensive profile of developing murine lens transcriptome including both mRNA and miRNA through next-generation RNA sequencing. A complete repository of the lens transcriptome of six developmental time points will be monumental in elucidating processes essential for development of the ocular lens and maintenance its transparency.
2015-10-29 | GSE69221 | GEO
Project description:Purpose: Transcriptome is the entire repertoire of all transcripts present in a cell at any particular time. We undertook next-generation whole transcriptome sequencing approach to gain insight of the transcriptional landscape of the developing mouse lens. Methods: We ascertained mice lenses at six developmental time points including two embryonic (E15 and E18) and four postnatal stages (P0, P3, P6, and P9). The ocular tissue at each time point was maintained as two distinct pools serving as biological replicates for each developmental stage. The mRNA and small RNA libraries were paired-end sequenced on Illumina HiSeq 2000 and subsequently analyzed using bioinformatics tools. Results: Mapping of mRNA and small RNA libraries generated 187.56 and 154.22 million paired-end reads, respectively. We detected a total of 14,465 genes in the mouse ocular lens. Of these, 46 genes exhibited 40-fold differential expression compared to transcriptional levels at E15. Likewise, small RNA profiling identified 379 microRNAs (miRNAs) expressed in mouse lens. Of these, 49 miRNAs manifested an 8-fold or higher differential expression when compared, as above to the microRNA expression at E15. Conclusion: We report the first comprehensive profile of developing murine lens transcriptome including both mRNA and miRNA through next-generation RNA sequencing. A complete repository of the lens transcriptome of six developmental time points will be monumental in elucidating processes essential for development of the ocular lens and maintenance its transparency. Whole transcrtiome and microRNA profilling of mouse lens using 2 embryonic (E15 and E18) and 4 postnatal stages (P0, P3, P6 and P9) in duplicates through high-throughput sequening using Illumina HiSeq2000.
2015-10-29 | E-GEOD-69221 | biostudies-arrayexpress
Project description:The vertebrate lens is a valuable model system for investigating the gene expression changes that coordinate tissue differentiation due to its inclusion of two spatially separated cell types, the outer epithelial cells and the deeper denucleated fiber cells that they support. Zebrafish are a useful model system for studying lens development given the organ's rapid development in the first several days of life in an accessible, transparent embryo. While we have strong foundational knowledge of the diverse lens crystallin proteins and the basic gene regulatory networks controlling lens development, no study has detailed gene expression in a vertebrate lens at single cell resolution. Here we report an atlas of lens gene expression in zebrafish embryos and larvae at single cell resolution through five days of development, identifying a number of novel putative regulators of lens development. Our data address open questions about the temperospatial expression of α-crystallins during lens development that will support future studies of their function and provide the first detailed view of β- and γ-crystallin expression in and outside the lens. We describe divergent expression in transcription factor genes that occur as paralog pairs in the zebrafish. Finally, we examine the expression dynamics of cytoskeletal, membrane associated, RNA-binding, and transcription factor genes, identifying a number of novel patterns. Overall these data provide a foundation for identifying and characterizing lens developmental regulatory mechanisms and revealing targets for future functional studies with potential therapeutic impact.
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Project description:IntroductionMeningiomas are the most common primary intracranial tumor. Recent next generation sequencing analyses have elaborated the molecular drivers of this disease. We aimed to identify and characterize novel fusion genes in meningiomas.MethodsWe performed a secondary analysis of our RNA sequencing data of 145 primary meningioma from 140 patients to detect fusion genes. Semi-quantitative rt-PCR was performed to confirm transcription of the fusion genes in the original tumors. Whole exome sequencing was performed to identify copy number variations within each tumor sample. Comparative RNA seq analysis was performed to assess the clonality of the fusion constructs within the tumor.ResultsWe detected six fusion events (NOTCH3-SETBP1, NF2-SPATA13, SLC6A3-AGBL3, PHF19-FOXP2 in two patients, and ITPK1-FBP2) in five out of 145 tumor samples. All but one event (NF2-SPATA13) led to extremely short reading frames, making these events de facto null alleles. Three of the five patients had a history of childhood radiation. Four out of six fusion events were detected in expression type C tumors, which represent the most aggressive meningioma. We validated the presence of the RNA transcripts in the tumor tissue by semi-quantitative RT PCR. All but the two PHF19-FOXP2 fusions demonstrated high degrees of clonality.ConclusionsFusion genes occur infrequently in meningiomas and are more likely to be found in tumors with greater degree of genomic instability (expression type C) or in patients with history of cranial irradiation.
| S-EPMC7553203 | biostudies-literature
Project description:BACKGROUND: Despite recent advances, the transcriptional hierarchy driving pancreas organogenesis remains largely unknown, in part due to the paucity of comprehensive analyses. To address this deficit we generated ten SAGE libraries from the developing murine pancreas spanning Theiler stages 17-26, making use of available Pdx1 enhanced green fluorescent protein (EGFP) and Neurog3 EGFP reporter strains, as well as tissue from adult islets and ducts. RESULTS: We used a specificity metric to identify 2,536 tags with pancreas-enriched expression compared to 195 other mouse SAGE libraries. We subsequently grouped co-expressed transcripts with differential expression during pancreas development using K-means clustering. We validated the clusters first using quantitative real time PCR and then by analyzing the Theiler stage 22 pancreas in situ hybridization staining patterns of over 600 of the identified genes using the GenePaint database. These were then categorized into one of the five expression domains within the developing pancreas. Based on these results we identified a cascade of transcriptional regulators expressed in the endocrine pancreas lineage and, from this, we developed a predictive regulatory network describing beta-cell development. CONCLUSION: Taken together, this work provides evidence that the SAGE libraries generated here are a valuable resource for continuing to elucidate the molecular mechanisms regulating pancreas development. Furthermore, our studies provide a comprehensive analysis of pancreas development, and insights into the regulatory networks driving this process are revealed.
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