Dataset Information

LC-MS/MS identification of proteins that were bound to recombinant His-tagged isoforms of RPL22L1

ABSTRACT: Glioblastoma (GBM) is characterized by an exceptionally high intratumoral heterogeneity. However, the molecular mechanisms underlying the origin of different GBM cell populations remain unclear. Here we found that the composition of ribosomes of GBM cells in the tumor core and edge differ due to alternative RNA splicing. The acidic pH in the core switches pre-mRNA splicing of the ribosomal gene RPL22L1 toward the RPL22L1b isoform. To elucidate the functions of RPL22L1 isoforms we identified proteins that interact with RPL22L1a and RPL22L1b. First, each isoform was expressed in E. coli and immobilized on magnetic beads. The beads were incubated with GBM cell lysates and proteins bound to the beads were identified using LC-MS/MS.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain, Primary Cell, Cell Culture

DISEASE(S): Glioblastoma

SUBMITTER: Georgij Arapidi

LAB HEAD: Georgij Arapidi

PROVIDER: PXD035849 | Pride | 2023-10-24

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 20

Publications

Alternative RNA splicing modulates ribosomal composition and determines the spatial phenotype of glioblastoma cells.

Larionova Tatyana D TD Bastola Soniya S Aksinina Tatiana E TE Anufrieva Ksenia S KS Wang Jia J Shender Victoria O VO Andreev Dmitriy E DE Kovalenko Tatiana F TF Arapidi Georgij P GP Shnaider Polina V PV Kazakova Anastasia N AN Latyshev Yaroslav A YA Tatarskiy Victor V VV Shtil Alexander A AA Moreau Pascale P Giraud Francis F Li Chaoxi C Wang Yichan Y Rubtsova Maria P MP Dontsova Olga A OA Condro Michael M Ellingson Benjamin M BM Shakhparonov Mikhail I MI Kornblum Harley I HI Nakano Ichiro I Pavlyukov Marat S MS

Nature cell biology 20221003 10

Glioblastoma (GBM) is characterized by exceptionally high intratumoral heterogeneity. However, the molecular mechanisms underlying the origin of different GBM cell populations remain unclear. Here, we found that the compositions of ribosomes of GBM cells in the tumour core and edge differ due to alternative RNA splicing. The acidic pH in the core switches before messenger RNA splicing of the ribosomal gene RPL22L1 towards the RPL22L1b isoform. This allows cells to survive acidosis, increases ste ...[more]

PMID: 36192632

Similar Datasets

Project description:We developed native elongating transcript sequencing (NET-seq, Churchman and Weissman Nature 2011, PMID: 21248844) combined with RNase footprinting of nascent transcripts (RNET-seq) to visualize translocation dynamics and nascent transcript errors in paused RNA polymerases in E. coli. We employed RNET-seq to the wild-type (WT) E. coli strain and to an isogenic strain deficient in genes for GreA and GreB (ΔgreAB). Gre factors and their eukaryotic analog TFIIS rescue backtracked complexes of RNAP. Briefly, the cells were rapidly lysed via spheroplasting, and the transcribing RNAPs were released from the genomic DNA by digestion with DNase I. Any ribosomes involved in co-transcriptional translation were separated from RNAP by digestion with RNase A. All RNAPs including those associated with the fragmented double-stranded DNAs and their 5’-truncated nascent RNAs were immobilized on Ni2+-NTA beads through the hexa-histidine-tagged β’ subunit and then extensively washed with a high-salt buffer. The 5’ ends of the transcripts in ECs were trimmed with RNase T1/V1 to leave a minimal length of RNA protected by RNAP. The RNases were subsequently removed by further washing of the beads. Elution with imidazole generated ECs carrying ~6-30 nt long transcripts. The nascent RNAs isolated from the ΔgreAB strain were longer than those from the WT strain and peaked at 18 nt versus 16 nt suggesting an enrichment of backtracked ECs, which is expected to occur in the absence of Gre-dependent 3’ RNA cleavage. The RNET-seq method involves trimming of excess nascent RNA from the 5’ ends leaving only the nascent RNA that is protected by RNAP. A previous in vitro study showed that an RNAP forming an EC protects different lengths of the 3’-proximal transcript from trimming by RNases A and T1 depending on the EC translocation state. Post-translocated, pre-translocated, and backtracked complexes protect 14-nt, 15-nt and >15-nt segments of the RNA, respectively. Because the very 3’ end of the RNA is extruded to a narrow pore within front of the enzyme during backtracking, the extruded RNA remains inaccessible to RNases increasing in length as backtracking increases. Thus, paused RNAP in either the pre- and post-translocated states as well as in different backtracked distances were monitored over the entire genome. The unique properties of our RNET-seq approach provided an opportunity to dissect the core mechanisms of different types of pausing in living cells.

			Action	DRS
	RPL22L1_2_Frozen.sf3	Other
	Sh-79.dat	Other
	Sh-79.mgf	Mgf
	Sh-79.mzid.gz	Mzid
	Sh-79.mzid_Sh-79.MGF	Mzid