Browse
Submit Data
Databases
API
Help

Dataset Information

171 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

ProteomeGenerator analysis of K052

ABSTRACT: To improve identification of canonical and non-canonical protein isoforms, we introduced ProteomeGenerator, a framework for reference-guided and de novo proteogenomic database generation from transcriptomic sequencing dataset. The proteomic databases output by ProteomeGenerator contain only proteins encoded by actively transcribed genes, and includes sample-specific protein isoforms resulting from non-canonical transcription and mRNA editing. We applied this workflow to the proteogenomic analysis of spliceosome-defective K052 SRSF2(P95H) cells, demonstrating high-confidence identification of proteins isoforms arising from intron inclusion and non-canonical splicing, as well as improved overall estimation of false-discovery rate from the focused database assembled by ProteomeGenerator.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Alex Kentsis

LAB HEAD: Alex Kentsis

PROVIDER: PXD008381 | Pride | 2018-10-11

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Publications

ProteomeGenerator: A Framework for Comprehensive Proteomics Based on de Novo Transcriptome Assembly and High-Accuracy Peptide Mass Spectral Matching.

Cifani Paolo P Dhabaria Avantika A Chen Zining Z Yoshimi Akihide A Kawaler Emily E Abdel-Wahab Omar O Poirier John T JT Kentsis Alex A

Journal of proteome research 20181019 11

Modern mass spectrometry now permits genome-scale and quantitative measurements of biological proteomes. However, analysis of specific specimens is currently hindered by the incomplete representation of biological variability of protein sequences in canonical reference proteomes and the technical demands for their construction. Here, we report ProteomeGenerator, a framework for de novo and reference-assisted proteogenomic database construction and analysis based on sample-specific transcriptome ...[more]

PMID: 30295032

Similar Datasets

Distinct and convergent consequences of splice factor mutations in myelodysplastic syndromes

Project description:Myelodysplastic syndromes (MDS) are characterized by recurrent somatic alterations often affecting components of RNA splicing machinery. Mutations of splice factors SF3B1, SRSF2, ZRSR2 and U2AF1 occur in >50% of MDS. To assess the impact of spliceosome mutations on splicing and to identify common pathways/genes affected by distinct mutations, we performed RNA-sequencing of 24 MDS bone marrow samples harboring spliceosome mutations (including hotspot alterations of SF3B1, SRSF2 and U2AF1; small deletions of SRSF2 and truncating mutations of ZRSR2), and devoid of other common co-occurring mutations. We uncover the landscape of splicing alterations in each splice factor mutant MDS and demonstrate that SRSF2 deletions cause highest number of splicing alterations compared with other spliceosome mutations. Although the mis-spliced events observed in different splice factor mutations were largely non-overlapping, a subset of genes, including EZH2, were aberrantly spliced in multiple mutant groups. Pathway analysis revealed that the mis-spliced genes in different mutant groups were enriched in RNA splicing and transport as well as several signaling cascades, suggesting converging biological consequences downstream of distinct spliceosome mutations.

2019-11-07 | GSE128429 | GEO

Spliceosome profiling visualizes the operations of a dynamic RNP in vivo at nucleotide resolution

Project description:Tools to understand how the spliceosome functions in vivo have lagged behind advances in its structural biology. We describe methods to globally profile spliceosome-bound precursor, intermediates and products at nucleotide resolution. We apply these tools to three divergent yeast species that span 600 million years of evolution. The sensitivity of the approach enables detection of novel cases of non- canonical catalysis including interrupted, recursive and nested splicing. Employing statistical modeling to understand the quantitative relationships between RNA features and the data, we uncover independent roles for intron size, position and number in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal ATP-dependent discard of numerous endogenous substrates at both the precursor and lariat-intermediate stages and connect discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology to investigate an RNP central to eukaryotic gene expression.

2018-02-19 | GSE108059 | GEO

Modulation of splicing catalysis for therapeutic targeting of leukemia with mutations in genes encoding spliceosomal proteins

Project description:Mutations in genes encoding splicing factors (which we refer to as spliceosomal genes) are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations recurrently affect specific amino acid residues, leading to perturbed normal splice site and exon recognition. Spliceosomal gene mutations are always heterozygous and rarely occur together with one another, suggesting that cells may tolerate only a partial deviation from normal splicing activity. To test this hypothesis, we engineered mice to express a mutated allele of serine/arginine-rich splicing factor 2 (Srsf2P95H) - which commonly occurs in individuals with MDS and AML - in an inducible, hemizygous manner in hematopoietic cells. These mice rapidly succumbed to fatal bone marrow failure, demonstrating that Srsf2-mutated cells depend on the wild-type Srsf2 allele for survival. In the context of leukemia, treatment with the spliceosome inhibitor E7107 resulted in substantial reductions in leukemic burden, specifically in isogenic mouse leukemias and patient-derived xenograft AMLs carrying spliceosomal mutations. Whereas E7107 treatment of mice resulted in widespread intron retention and cassette exon-skipping in leukemic cells regardless of Srsf2 genotype, the magnitude of splicing inhibition following E7107 treatment was greater in Srsf2-mutated than in Srsf2-wild-type leukemia, consistent with the differential effect of E7107 on survival. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal gene mutations are preferentially susceptible to additional splicing perturbations in vivo as compared to leukemias without such mutations. Modulation of spliceosome function may thus provide a new therapeutic avenue in genetically defined subsets of individuals with MDS or AML.

2016-03-31 | GSE74064 | GEO

The major and minor spliceosome interact to regulate alternative splicing [PBMCs]

Project description:Mutations in minor spliceosome components are linked to diseases such as Roifman syndrome, Lowry-Wood syndrome, and early-onset cerebellar ataxia (EOCA). Here we report that besides increased minor intron retention, Roifman syndrome and EOCA can also be characterized by elevated alternative splicing (AS) around minor introns. Consistent with the idea that the assembly/activity of the minor spliceosome informs AS in minor intron-containing genes (MIGs), inhibition of all minor spliceosome snRNAs led to upregulated AS. Notably, alternatively spliced MIG isoforms were bound to polysomes in the U11-null dorsal telencephalon, which suggested that aberrant MIG protein expression could contribute to disease pathogenesis. In agreement, expression of an aberrant isoform of the MIG Dctn3 by in utero electroporation, affected radial glial cell divisions. Finally, we show that AS around minor introns is executed by the major spliceosome and is regulated by U11-59K of the minor spliceosome, which forms exon-bridging interactions with proteins of the major spliceosome. Overall, we extend the exon-definition model to MIGs and postulate that disruptions of exon-bridging interactions might contribute to disease severity and pathogenesis.

2021-05-01 | GSE151141 | GEO

The major and minor spliceosome interact to regulate alternative splicing [HEK293 cells]

2021-05-01 | GSE151140 | GEO

RNA sequencing for human induced pluripotent stem cell cardiomyocyte differentiation

Project description:We apply short-read RNA sequencing technology to identify transcripts expressed during four time points of a human induced pluripotent stem cell derived cardiomyocyte differentiation protocol, corresponding to pluripotent, mesoderm, early cardiomyocyte, and cardiomyocyte cell stages. The RNA-seq reads are used to generate custom protein sequence database for proteogenomic applications and downstream mass spectrometry analysis. We demonstrate that this custom RNA-seq-guided proteomics approach can be used to identify protein isoforms that are differentially regulated during cardiac differentiation.

2019-11-15 | GSE137920 | GEO

CDK11 phosphorylates SF3B1 and is essential for constitutive splicing (RNA-seq)

Project description:Pre-mRNA splicing is a highly regulated process catalyzing intron excision by spliceosome. Spliceosome activation is a major control step requiring dramatic protein and RNA rearrangements leading to a catalytically active complex. Prior research has linked hyperphosphorylation of SF3B1, a subunit of U2 snRNP, with spliceosome activation and catalytically active spliceosome, rendering a relevant kinase a key player for pre-mRNA splicing. Here we use OTS964, the first potent inhibitor of cyclin-dependent kinase 11 (CDK11), to show rapid and selective dephosphorylation of SF3B1 on threonines required for spliceosome activation. CDK11 associates with SF3B1 and its inhibition causes massive intron retention, block in precatalytic spliceosome complex B to activated spliceosome complex Bact transition and accumulation of non-functional spliceosomes on pre-mRNA and chromatin. These studies reveal crucial regulatory role of CDK11 in human pre-mRNA splicing and define the compound OTS964 as a quality chemical biology probe for CDK11.

2022-07-09 | GSE185809 | GEO