TY - JOUR

T1 - Tetrapeptide 60-63 of human ribosomal protein uS3 is crucial for translation initiation

AU - Babaylova, Elena

AU - Malygin, Alexey

AU - Gopanenko, Alexander

AU - Graifer, Dmitri

AU - Karpova, Galina

N1 - Publisher Copyright: © 2019 Elsevier B.V.

PY - 2019/9/1

Y1 - 2019/9/1

N2 - Conserved ribosomal protein uS3 contains a decapeptide fragment in positions 55-64 (human numbering), which has a very specific ability to cross-link to various RNA derivatives bearing aldehyde groups, likely provided by K62. It has been shown that during translation in the cell-free protein-synthesizing system, uS3 becomes accessible for such cross-linking only after eIF3j leaves the mRNA binding channel of the 40S ribosomal subunit. We studied the functional role of K62 and its nearest neighbors in the ribosomal assembly and translation with the use of HEK293T-derived cell cultures capable of producing FLAG-tagged uS3 (uS3FLAG) or its mutant form with amino acid residues at positions 60-63 replaced with alanines. Analysis of polysome profiles from the respective cells and cytosol lysates showed that the mutation significantly affected the uS3 ability to participate in the assembly of 40S subunits, but it was not essential for their maturation and did not prevent the binding of mRNAs to 40S subunits during translation initiation. The most striking effect of the replacement of amino acid residues in the above uS3 positions was that it almost completely deprived the 40S subunits of their ability to form 80S ribosomes, suggesting that the 48S pre-initiation complexes assembled on these subunits were defective in the binding of 60S subunits. Thus, our results revealed the previously unknown crucial role of the uS3 tetrapeptide 60GEKG63 in translation initiation related to maintaining the proper structure of the 48S complex, most likely via the prevention of premature mRNA loading into the ribosomal channel.

AB - Conserved ribosomal protein uS3 contains a decapeptide fragment in positions 55-64 (human numbering), which has a very specific ability to cross-link to various RNA derivatives bearing aldehyde groups, likely provided by K62. It has been shown that during translation in the cell-free protein-synthesizing system, uS3 becomes accessible for such cross-linking only after eIF3j leaves the mRNA binding channel of the 40S ribosomal subunit. We studied the functional role of K62 and its nearest neighbors in the ribosomal assembly and translation with the use of HEK293T-derived cell cultures capable of producing FLAG-tagged uS3 (uS3FLAG) or its mutant form with amino acid residues at positions 60-63 replaced with alanines. Analysis of polysome profiles from the respective cells and cytosol lysates showed that the mutation significantly affected the uS3 ability to participate in the assembly of 40S subunits, but it was not essential for their maturation and did not prevent the binding of mRNAs to 40S subunits during translation initiation. The most striking effect of the replacement of amino acid residues in the above uS3 positions was that it almost completely deprived the 40S subunits of their ability to form 80S ribosomes, suggesting that the 48S pre-initiation complexes assembled on these subunits were defective in the binding of 60S subunits. Thus, our results revealed the previously unknown crucial role of the uS3 tetrapeptide 60GEKG63 in translation initiation related to maintaining the proper structure of the 48S complex, most likely via the prevention of premature mRNA loading into the ribosomal channel.

KW - Amino acid replacements

KW - KH domain

KW - Mammalian ribosomal protein uS3

KW - Pre-initiation complexes

KW - Small ribosomal subunit maturation

KW - Translation initiation

KW - S3

KW - EIF3

KW - STATES

KW - MESSENGER-RNA

KW - ENTRY CHANNEL

UR - http://www.scopus.com/inward/record.url?scp=85071897240&partnerID=8YFLogxK

U2 - 10.1016/j.bbagrm.2019.194411

DO - 10.1016/j.bbagrm.2019.194411

M3 - Article

C2 - 31356988

AN - SCOPUS:85071897240

VL - 1862

SP - 194411

JO - Biochimica et Biophysica Acta - Gene Regulatory Mechanisms

JF - Biochimica et Biophysica Acta - Gene Regulatory Mechanisms

SN - 1874-9399

IS - 9

M1 - 194411

ER -