PXD019034-2

PXD019034 is an original dataset announced via ProteomeXchange.

Title	Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia
Description	To gain functional insights into human TSEN/CLP1, we designed an expression vector series based on the MultiBac system that allows combinatorial protein complex production in insect and mammalian cells by utilizing a CMV/p10 dual promoter. Using this system, we were able to assemble and purify functional heterotetrameric TSEN and a heteropentameric complex including the RNA kinase CLP1 from infected insect cells. Individual proteins of the complexes were identified using in-gel digestion and LC-MS/MS analysis. To gain atomistic insights into the molecular architecture of the human TSEN complex, we set out to characterize the TSEN15–34 heterodimer by X-ray crystallography. Despite extensive crystallization trials, full-length TSEN15–34 did not yield any crystals. To define a crystallizable core complex, we subjected the full-length complex to limited proteolysis with subsequent size exclusion chromatography. Primarily, we observed two comigrating polypeptide species. The two polypeptide species were identified using in-gel digestion followed by LC-MS/MS analysis. This led to the identification of the polypeptide species corresponding to residues 23 to 170 of TSEN15 and residues 208 to 310 of TSEN34 covering the predicted conserved nuclease domains.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_05:23:02.717.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Marie Alfes
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2020-05-07 01:20:28	ID requested
1	2021-06-16 23:02:34	announced
⏵ 2	2024-10-22 05:23:03	announced	2024-10-22: Updated project metadata.

Dataset with its publication pending

submitter keyword: TSEN, LC-MS/MS

Carla Schmidt
contact affiliation	IWE ZIK HALOmem, Martin Luther University Halle-Wittenberg, Halle, Germany
contact email	carla.schmidt@biochemtech.uni-halle.de
lab head
Marie Alfes
contact affiliation	HALOmem, Martin Luther University Halle-Wittenberg
contact email	marie.barth@bct.uni-halle.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD019034
     1. Label: PRIDE project
     2. Name: Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia