Adapted formaldehyde gradient cross-linking protocol implicates human eIF3d and eIF3c, k and l subunits in the 43S and 48S pre-initiation complex assembly, respectively.
Cross-Linking Reagents
/ pharmacology
Eukaryotic Initiation Factor-3
/ genetics
Formaldehyde
/ pharmacology
Humans
Microtubule-Associated Proteins
/ genetics
Protein Binding
Protein Biosynthesis
/ genetics
RNA, Messenger
/ genetics
Ribosome Subunits, Small, Eukaryotic
/ genetics
Ribosomes
/ genetics
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
28 02 2020
28 02 2020
Historique:
accepted:
09
12
2019
revised:
04
12
2019
received:
04
11
2019
pubmed:
22
12
2019
medline:
19
3
2020
entrez:
22
12
2019
Statut:
ppublish
Résumé
One of the key roles of the 12-subunit eukaryotic translation initiation factor 3 (eIF3) is to promote the formation of the 43S and 48S pre-initiation complexes (PICs). However, particular contributions of its individual subunits to these two critical initiation reactions remained obscure. Here, we adapted formaldehyde gradient cross-linking protocol to translation studies and investigated the efficiency of the 43S and 48S PIC assembly in knockdowns of individual subunits of human eIF3 known to produce various partial subcomplexes. We revealed that eIF3d constitutes an important intermolecular bridge between eIF3 and the 40S subunit as its elimination from the eIF3 holocomplex severely compromised the 43S PIC assembly. Similarly, subunits eIF3a, c and e were found to represent an important binding force driving eIF3 binding to the 40S subunit. In addition, we demonstrated that eIF3c, and eIF3k and l subunits alter the efficiency of mRNA recruitment to 43S PICs in an opposite manner. Whereas the eIF3c knockdown reduces it, downregulation of eIF3k or eIF3l increases mRNA recruitment, suggesting that the latter subunits possess a regulatory potential. Altogether this study provides new insights into the role of human eIF3 in the initial assembly steps of the translational machinery.
Identifiants
pubmed: 31863585
pii: 5682903
doi: 10.1093/nar/gkz1185
pmc: PMC7039009
doi:
Substances chimiques
Cross-Linking Reagents
0
EIF3C protein, human
0
EIF3D protein, human
0
EIF3K protein, human
0
EIF3L protein, human
0
Eukaryotic Initiation Factor-3
0
Microtubule-Associated Proteins
0
RNA, Messenger
0
Formaldehyde
1HG84L3525
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1969-1984Subventions
Organisme : Wellcome Trust
ID : 090812/B/09/Z
Pays : United Kingdom
Informations de copyright
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
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