Cell-specific translational regulation of S-adenosylmethionine decarboxylase mRNA. Influence of the structure of the 5' transcript leader on regulation by the upstream open reading frame.

H Ruan,J.R Hill,S Fatemie-Nainie,D.R Morris

doi:10.1016/s0021-9258(17)32395-5

H Ruan, J.R Hill + Show 2 more

Open Access

https://doi.org/10.1016/s0021-9258(17)32395-5

Copy DOI

Abstract

A small upstream open reading frame (uORF), located 14 nucleotides from the cap in the 5' transcript leader (5' TL) of the mRNA encoding S-adenosylmethionine decarboxylase (AdoMetDC), suppresses translation of the downstream cistron in normal T cells and T cell lines. In the present study, we examined the structural features of the 5' TL that overcome this suppressive influence in cells of nonlymphoid origin. Initiation at the downstream cistron in nonlymphoid cells is by a cap-dependent mechanism that requires ribosome scanning along the 5' TL and does not involve an internal ribosome entry site. Extending the uORF so that it overlapped the major cistron by 101 nucleotides had no effect on translation of the downstream cistron in either HeLa or Jurkat cells. When the distance between the uORF and the cap was extended to 47 nucleotides, using sequence previously found to be neutral, translation of the major cistron was inhibited 5-fold in HeLa cells and the mRNA was moved from polysomes to monosomes, a location identical to that of the wild type mRNA in Jurkat cells. Therefore, in contrast to T cells, initiation at the uORF seems to be relatively infrequent in non-lymphoid cells due to its proximity to the cap, allowing efficient translation of the downstream cistron.

Highlights

INFLUENCE OF THE STRUCTURE OF THE 5’ TRANSCRIPT LEADER ON REGULATION BY THE UPSTREAM OPEN READING FRAME*
The results are consistent waitmh odel tion is the presence of initiation codons or upstreamopen read- where theuORF is ignored in non-lymphoid cells because of its ing frames within the 5’ TL of a particular mRNA. proximity to the 5‘ end of the mRNA and, as a consequence, These sequences can dramatically suppress translationof the allowing efficient translation of the downstream cistron
The 5' leader of the AdoMetDC mRNAcontains a long inter- only 10% of AdoMetDC mRNA remained on polysomes at 4 h cistronic region of slightly less than 300 nucleotides between post-infection

Summary

EXPERIMENTAL PROCEDURES

Cell Culture-The Jurkat (E-6)cell line was provided by R. S1 Nuclease Protection Assay-The structures of the 5' ends of the chimeric constructs were determined usingmaodified S1 nuclease procodon of the uORF (Fig. 1).We inserted an inverted repeat, with the potentiaolf forming a stable hairpin(AG = -61 kcallmol) in theRNA transcript, into thBe gZII site t o generate construct pRSSL390 (Fig. 1).The identicalstem-loop sequence was shown previously to block scanning of the 40 S ribosomal subunit and inhibit cap-dependent translation (Kozak, tection assay (Greene and Struhl, 1988) as described previously (Hill 1989) We transfected these constructs into HeLa cells and and Morris, 1992).Briefly, constructs pRS327 and pRS362 were transfected into HeLa cells.

RESULTS

Translation efficiency”

Relative manslation rffiriencv

There aretwo important aspectsof regulation of translation