Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

Schmidt, Carla; Grønborg, Mads; Deckert, Jochen; Bessonov, Sergey; Conrad, Thomas; Lührmann, Reinhard; Urlaub, Henning

doi:10.1261/rna.041244.113

Cited by 72 publications

(77 citation statements)

References 44 publications

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“…Furthermore, the assembly pathway in vitro is characterized by major changes in composition (e.g., only three of the five spliceosomal snRNPs are part of the active C complex [36]). On the other hand, we have previously shown that both the general population of supraspliceosomes and native spliceosomes contained all five spliceosomal snRNPs [15].…”

Section: Resultsmentioning

confidence: 99%

Supraspliceosomes at Defined Functional States Portray the Pre-Assembled Nature of the Pre-mRNA Processing Machine in the Cell Nucleus

Kotzer-Nevo

Alves

Rappsilber

et al. 2014

IJMS

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When isolated from mammalian cell nuclei, all nuclear pre-mRNAs are packaged in multi-subunit large ribonucleoprotein complexes—supraspliceosomes—composed of four native spliceosomes interconnected by the pre-mRNA. Supraspliceosomes contain all five spliceosomal U snRNPs, together with other splicing factors, and are functional in splicing. Supraspliceosomes studied thus far represent the steady-state population of nuclear pre-mRNAs that were isolated at different stages of the splicing reaction. To analyze specific splicing complexes, here, we affinity purified Pseudomonas aeruginosa phage 7 (PP7)-tagged splicing complexes assembled in vivo on Adenovirus Major Late (AdML) transcripts at specific functional stages, and characterized them using molecular techniques including mass spectrometry. First, we show that these affinity purified splicing complexes assembled on PP7-tagged AdML mRNA or on PP7-tagged AdML pre-mRNA are assembled in supraspliceosomes. Second, similar to the general population of supraspliceosomes, these defined supraspliceosomes populations are assembled with all five U snRNPs at all splicing stages. This study shows that dynamic changes in base-pairing interactions of U snRNA:U snRNA and U snRNA:pre-mRNA that occur in vivo during the splicing reaction do not require changes in U snRNP composition of the supraspliceosome. Furthermore, there is no need to reassemble a native spliceosome for the splicing of each intron, and rearrangements of the interactions will suffice.

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Section: Resultsmentioning

confidence: 99%

Supraspliceosomes at Defined Functional States Portray the Pre-Assembled Nature of the Pre-mRNA Processing Machine in the Cell Nucleus

Kotzer-Nevo

Alves

Rappsilber

et al. 2014

IJMS

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show abstract

“…phosphorylation, acetylation, ubiquitination) (90). Furthermore, this technique was already successfully used to identify many novel RNA-binding proteins that interact with various classes of RNAs, such as pri- or pre- miRNAs (30), telomeric repeat-containing RNA (91), spliceosomal snRNAs (92,93), viral RNAs (61,76) and expanded GGGGCC hexanucleotide repeats (94). …”

Section: Proteomic Analysis Of Isolated Rbpsmentioning

confidence: 99%

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

Jazurek¹,

Ciesiolka²,

Staręga-Rosłan³

et al. 2016

Nucleic Acids Res

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RNA–protein complexes play a central role in the regulation of fundamental cellular processes, such as mRNA splicing, localization, translation and degradation. The misregulation of these interactions can cause a variety of human diseases, including cancer and neurodegenerative disorders. Recently, many strategies have been developed to comprehensively analyze these complex and highly dynamic RNA–protein networks. Extensive efforts have been made to purify in vivo-assembled RNA–protein complexes. In this review, we focused on commonly used RNA-centric approaches that involve mass spectrometry, which are powerful tools for identifying proteins bound to a given RNA. We present various RNA capture strategies that primarily depend on whether the RNA of interest is modified. Moreover, we briefly discuss the advantages and limitations of in vitro and in vivo approaches. Furthermore, we describe recent advances in quantitative proteomics as well as the methods that are most commonly used to validate robust mass spectrometry data. Finally, we present approaches that have successfully identified expanded repeat-binding proteins, which present abnormal RNA–protein interactions that result in the development of many neurological diseases.

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“…Comparisons across different samples were performed after normalization of the total spectra. Spectral counting was used to compare protein abundances at different time points according to a previously described procedure [15,16].…”

Section: Database Searching and Label-free Quantitationmentioning

confidence: 99%

Early urinary protein changes in the tumor-forming process of the NuTu-19 lung metastasis

Wei

Meng

et al. 2019

Preprint

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The early detection of cancer is essential for effective intervention. Urine, which lacks homeostatic control, has been used to reflect early systemic changes in subcutaneous cancer models. However, urine has not been used to predict whether tumors will be formed in animal models. In this study, a cancer model was established by the tail-vein injection of 2 million NuTu-19 tumor cells. Approximately half of the rats formed lung metastatic carcinoma tumors. Urine samples were randomly selected from 4 tumor-forming rats and 4 non-tumor-forming rats on day 0/12/27/39/52 and analyzed by label-free proteomic quantitative analysis. Compared to day 0, a total of 180 and 118 urinary proteins in tumor-forming and non-tumor-forming rats, respectively, showed significant changes. Functional enrichment analysis of the differential proteins in tumor-forming rats revealed similar events during cancer metastasis cascades and tumor progression, such as the migration of tumor cell lines, coagulation system, TGF-β signaling, the STAT3 pathway and the adhesion of myeloid cells and alveolar macrophages. The differential proteins in non-tumor-forming rats were associated with agranulocyte/granulocyte adhesion and diapedesis, glutathione biosynthesis, IL-12 signaling and vitamin metabolism. After validation by parallel reaction monitoring (PRM) targeted proteomics quantitative analysis, a total of 22 urinary proteins showed significant changes in the early phase of the lung tumor formation process, 5 of which have been associated with the mechanisms of lung cancer, while 3 have been suggested as lung cancer biomarkers. Another 14 urinary proteins showed significant changes in the early phase in non-tumor-forming rats. Our results indicate that urine proteins could differentiate early tumor-forming and non-tumor-forming status.

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Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

Cited by 72 publications

References 44 publications

Supraspliceosomes at Defined Functional States Portray the Pre-Assembled Nature of the Pre-mRNA Processing Machine in the Cell Nucleus

Supraspliceosomes at Defined Functional States Portray the Pre-Assembled Nature of the Pre-mRNA Processing Machine in the Cell Nucleus

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

Early urinary protein changes in the tumor-forming process of the NuTu-19 lung metastasis

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