Mass spectrometric analysis of the human 40S ribosomal subunit

Native and HCV IRES-bound complexes

Yonghao Yu, Hong Ji, Jennifer A. Doudna, Julie A. Leary

Research output: Contribution to journalArticle

69 Citations (Scopus)

Abstract

Hepatitis C virus uses an internal ribosome entry site (IRES) in the viral RNA to directly recruit human 40S ribosome subunits during cap-independent translation initiation. Although IRES-mediated translation initiation is not subject to many of the regulatory mechanisms that control cap-dependent translation initiation, it is unknown whether other noncanonical protein factors are involved in this process. Thus, a global protein composition analysis of native and IRES-bound 40S ribosomal complexes has been conducted to facilitate an understanding of the IRES ribosome recruitment mechanism. A combined top-down and bottom-up mass spectrometry approach was used to identify both the proteins and their posttranslational modifications (PTMs) in the native 40S subunit and the IRES recruited translation initiation complex. Thirty-one out of a possible 32 ribosomal proteins were identified by combining top-down and bottom-up mass spectrometry techniques. Proteins were found to contain PTMs, including loss of methionine, acetylation, methylation, and disulfide bond formation. In addition to the 40S ribosomal proteins, RACK1 was consistently identified in the 40S fraction, indicating that this protein is associated with the 40S subunit. Similar methodology was then applied to the hepatitis C virus IRES-bound 40S complex. Two 40S ribosomal proteins, RS25 and RS29, were found to contain different PTMs than those in the native 40S subunit. In addition, RACK1, eukaryotic initiation factor 3 proteins and nucleolin were identified in the IRES-mediated translation initiation complex.

Original languageEnglish (US)
Pages (from-to)1438-1446
Number of pages9
JournalProtein Science
Volume14
Issue number6
DOIs
StatePublished - Jun 2005

Fingerprint

Eukaryotic Small Ribosome Subunits
Ribosomal Proteins
Post Translational Protein Processing
Proteins
Viruses
Mass spectrometry
Eukaryotic Initiation Factor-3
Hepacivirus
Mass Spectrometry
Acetylation
Methylation
Viral RNA
Disulfides
Methionine
Internal Ribosome Entry Sites
Ribosomes
Chemical analysis

Keywords

  • Mass spectrometry
  • Protein mapping
  • Ribosome

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mass spectrometric analysis of the human 40S ribosomal subunit : Native and HCV IRES-bound complexes. / Yu, Yonghao; Ji, Hong; Doudna, Jennifer A.; Leary, Julie A.

In: Protein Science, Vol. 14, No. 6, 06.2005, p. 1438-1446.

Research output: Contribution to journalArticle

Yu, Yonghao ; Ji, Hong ; Doudna, Jennifer A. ; Leary, Julie A. / Mass spectrometric analysis of the human 40S ribosomal subunit : Native and HCV IRES-bound complexes. In: Protein Science. 2005 ; Vol. 14, No. 6. pp. 1438-1446.
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