Translational Control of the Hematopoiesis-Specific RhoH Gene Is Mediated by Upstream Open Reading Frames.

The hematopoiesis-specific RhoH gene was identified in our laboratory, as a BCL6 partner gene (1), in follicular B-non Hodgkin’s lymphoma associated with the recurring t(3;4) chromosomal translocation (2). The RhoH gene encodes a Rho small G protein supposed to be a Rac antagonist (3). The RhoH protein is always in a GTP-bound state (i.e. constitutively active), which suggests a strongly regulated synthesis. This work is aimed at elucidating molecular mechanisms that may regulate this RhoH protein synthesis.

The RhoH gene contains one coding exon only (exon 2) preceded by six uncoding exons, and the RhoH transcripts exhibit an important 5′ UTR heterogeneity, especially in the B-lymphoid lineage. This heterogeneity both reflects (i) an use of multiple transcription start sites and (ii) alternative splicing events of some 5′ uncoding exons (4). The present study is devoted to assess the functional relevance of these 5′ uncoding exons in the RhoH protein synthesis. Some of them (exons 1a, 1b and X4) contain an upstream ORF (uORF) sequence. Ten percent of eukaryotic mRNAs, 2/3 of which are involved in the control of cellular growth and differentiation, contain such uORFs within their 5′-UTR sequence. These uORFs are involved in the translational control of these genes (5). RhoH thus provides an additional opportunity to assess the role of uORFs in the translation of a cellular mRNA. Moreover, uORFs have never been implicated in the regulation of any small G family member gene.

The data show that the uORFs sequences from the RhoH gene repress the translation of a downstream reporter gene, in transfected hematopoietic cell lines (erythroid, B- and T- lymphoid). The initiation codon from 1a-, 1b-, and X4- uORFs is strongly involved in this phenomenon, as the inhibitory effect was suppressed by an uAUG invalidation. Targeted mutations (premature Stop, silent or mis-sens) within these uORFs sequences were functionally analyzed in the same reporter LUC system. The results led us to distinguish two uORF-mediated translational regulatory mechanisms: (i) an “uAUG dependent / uORF-peptide dependent” mechanism for the 1a- and 1b- uORFs sequences, and (ii) an “uAUG dependent / uORF-peptide independent” mechanism for the X4- uORF.

The RhoH transcripts exhibit a 5′ UTR heterogeneity, which might modulate the protein synthesis. Three main RhoH transcripts are expressed in the different hematopoietic lineages: “1a-X4-2”, “1a-1b-X4-2” and “1b-X4-2” (4), therefore the effects of the 1a-, 1b-, and X4- uORFs were investigated in combination (1a+X4; 1a+1b+X4; 1b+X4), as in the RhoH transcripts. The results convinced us to propose an uORF-mediated translational regulatory mechanism, which is based on a combinatory effect (synergy or antagonism) of three uORFs. This mechanism is called: “translational lock-unlock” model of the RhoH gene. In vitro translation experiments aimed at validating this model will be presented.