Global and specific translational control by rapamycin in T cells uncovered by microarrays and proteomics.
Rapamycin has been shown to affect translation. We have utilized two complementary approaches to identify genes that are predominantly affected by rapamycin in Jurkat T cells. One was to compare levels of polysome-bound and total RNA using oligonucleotide microarrays complementary to 6,300 human genes. Another was to determine protein synthesis levels using two-dimensional PAGE. Analysis of expression changes at the polysome-bound RNA levels showed that translation of most of the expressed genes was partially reduced following rapamycin treatment. However, translation of 136 genes (6% of the expressed genes) was totally inhibited. This group included genes encoding RNA-binding proteins and several proteasome subunit members. Translation of a set of 159 genes (7%) was largely unaffected by rapamycin treatment. These genes included transcription factors, kinases, phosphatases, and members of the RAS superfamily. Analysis of [(35)S]methionine-labeled proteins from the same cell populations using two-dimensional PAGE showed that the integrated intensity of 111 of 830 protein spots changed in rapamycin-treated cells by at least 3-fold (70 increased, 41 decreased). We identified 22 affected protein spots representing protein products of 16 genes. The combined microarray and proteomic approach has uncovered novel genes affected by rapamycin that may be involved in its immunosuppressive effect and other genes that are not affected at the level of translation in a context of general inhibition of cap-dependent translation.
Grolleau
Annabelle
A
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Bowman
Jessica
J
Pradet-Balade
Bérengère
B
Puravs
Eric
E
Hanash
Samir
S
Garcia-Sanz
Jose A
JA
Beretta
Laura
L
D015153:Blotting, Western; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D005786:Gene Expression Regulation; D006801:Humans; D019169:Jurkat Cells; D020411:Oligonucleotide Array Sequence Analysis; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism*; D013997:Time Factors; D015854:Up-Regulation
D012333:RNA, Messenger; D012313:RNA; D020123:Sirolimus