TY - JOUR

T1 - The eS26 protein is involved in the formation of a nucleophosmin binding site on the human 40S ribosomal subunit

AU - Ivanov, Anton V.

AU - Gopanenko, Alexander V.

AU - Malygin, Alexey A.

AU - Karpova, Galina G.

N1 - Copyright © 2018 Elsevier B.V. All rights reserved.

PY - 2018/5/1

Y1 - 2018/5/1

N2 - Human ribosomal protein eS26 is an indispensable component of the small (40S) ribosomal subunit and, along with other ribosomal proteins, is involved in interaction with mRNAs during translation. Here, we explored the behavior of the exogenous ribosomal protein eS26 modified at the C-terminus in the events related to translation in human cells using a doxycycline-inducible HEK293-derived cell line enabling the stable production of C-terminal FLAG-tagged eS26 (eS26FLAG). The production of eS26FLAG in cells was accompanied by a decrease in the endogenous eS26 content although its mRNA level did not change. Exogenous eS26FLAG was able to replace endogenous eS26 in 40S ribosomal subunits, without affecting the assembly and translational activity of 80S ribosomes. However, eS26FLAG-containing ribosome fractions from the respective polysome profile displayed a reduced content of nucleophosmin, a multifunctional protein, which, as is known, is involved in the formation and nuclear export of ribosomal subunits. In general, our data showed that although the appearance of the FLAG tag at the C-terminus of eS26 does not affect translation, it interferes with nucleophosmin incorporation into the 40S subunit, pointing out the importance of the C-terminus integrity of eS26 for nucleophosmin binding. In addition, with the recombinant protein, we demonstrated the binding of nucleophosmin to both isolated eS26 and 40S subunits in the presence of HeLa nuclear extract that phosphorylated the recombinant nucleophosmin. These findings suggest that for nuclear export, nucleophosmin could directly bind to pre-40S subunits in the mRNA exit site region where the C-terminus of eS26 is located.

AB - Human ribosomal protein eS26 is an indispensable component of the small (40S) ribosomal subunit and, along with other ribosomal proteins, is involved in interaction with mRNAs during translation. Here, we explored the behavior of the exogenous ribosomal protein eS26 modified at the C-terminus in the events related to translation in human cells using a doxycycline-inducible HEK293-derived cell line enabling the stable production of C-terminal FLAG-tagged eS26 (eS26FLAG). The production of eS26FLAG in cells was accompanied by a decrease in the endogenous eS26 content although its mRNA level did not change. Exogenous eS26FLAG was able to replace endogenous eS26 in 40S ribosomal subunits, without affecting the assembly and translational activity of 80S ribosomes. However, eS26FLAG-containing ribosome fractions from the respective polysome profile displayed a reduced content of nucleophosmin, a multifunctional protein, which, as is known, is involved in the formation and nuclear export of ribosomal subunits. In general, our data showed that although the appearance of the FLAG tag at the C-terminus of eS26 does not affect translation, it interferes with nucleophosmin incorporation into the 40S subunit, pointing out the importance of the C-terminus integrity of eS26 for nucleophosmin binding. In addition, with the recombinant protein, we demonstrated the binding of nucleophosmin to both isolated eS26 and 40S subunits in the presence of HeLa nuclear extract that phosphorylated the recombinant nucleophosmin. These findings suggest that for nuclear export, nucleophosmin could directly bind to pre-40S subunits in the mRNA exit site region where the C-terminus of eS26 is located.

KW - HEK293 cells

KW - Human ribosome

KW - Nucleophosmin

KW - Protein C-terminal FLAG-tag

KW - Ribosomal protein eS26

KW - Nuclear Proteins/chemistry

KW - Protein Biosynthesis

KW - Humans

KW - Models, Molecular

KW - RNA, Messenger/genetics

KW - Transfection

KW - Ribosomes/chemistry

KW - HEK293 Cells

KW - Ribosomal Proteins/chemistry

KW - Protein Binding

KW - Protein Conformation

KW - HeLa Cells

KW - Ribosome Subunits, Small, Eukaryotic/chemistry

KW - Binding Sites

UR - http://www.scopus.com/inward/record.url?scp=85044597400&partnerID=8YFLogxK

U2 - 10.1016/j.bbapap.2018.03.004

DO - 10.1016/j.bbapap.2018.03.004

M3 - Article

C2 - 29563070

AN - SCOPUS:85044597400

VL - 1866

SP - 642

EP - 650

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 5-6

ER -