Signs of translational regulation within the transcript leader of a plant plasma membrane H⁺‐ATPase gene

Abstract

SummaryTranscripts of most plant plasma membrane H ⍣ -ATPase genes possess a leader (5Ј untranslated region) that is unusually long and that contains a short upstream open reading frame (uORF), two features which suggest posttranscriptional regulation. To investigate the putative role of the transcript leader, we have placed the leader of pma3, one of the Nicotiana plumbaginifolia H ⍣ -ATPase genes, between the CaMV 35S promoter and the sequence coding for the β-glucuronidase (GUS) reporter gene. Transient expression of this chimeric gene and of derived mutants was analysed in electroporated tobacco protoplasts. The whole leader had a positive effect on translation, since deletion of most of its sequence reduced GUS activity. Suppression of the uORF by point mutation of its initiating AUG increased GUS activity by about 55%. Analysis of various deletions and mutations suggested that the uORF is translated by at least twothirds of scanning ribosomes, half of which subsequently reinitiate downstream translation under our experimental conditions. Reinitiation did not depend on the nucleotide sequence of the uORF, nor on that separating the uORF and the main open reading frame. We conclude that the pma3 transcript possesses features of translational regulation, whose mode of functioning has yet to be discovered.

Identification of a pollen‐specific sucrose transporter‐like proteinNtSUT3 from tobacco

“…NtSUT3 shares 64.9% identity at the amino acid level with NtSUT1, and has all characteristics of plant sucrose transporters. Several attempts to demonstrate a sucrose transporter function in yeast failed and this could be related to features specific for open reading frame led to a 55% increase in translation in tobacco protoplasts [28]. Furthermore, NtSUT3 contains three in frame ATGs which might be detrimental to heterologous expression in yeast [29].…”

Translational Regulation of Cytoplasmic mRNAs

“…Upstream ORFs must be translated regularly, since AUG triplets in any context can be recognized by the scanning ribosome most of the time (Lukaszewicz et al, 1998;Sugio et al, 2010), and recent ribosome footprinting studies in yeast and metazoan mRNAs have proven that existing uORFs regularly engage ribosomes, sometimes even initiating on non-AUG triplets (Ingolia et al, 2009;Ingolia et al, 2011). Non-AUG initiation is probably fairly common in plants as well (Gordon et al, 1992;Kobayashi et al, 2002).…”

Targeting of a Nicotiana plumbaginifolia H+-ATPase to the Plasma Membrane Is Not by Default and Requires Cytosolic Structural Determinants

“…Protoplasts were prepared from N. tabacum leaves and transiently transformed by electroporation as described by Lukaszewicz et al (1998) using 6.45 pmol of each of the appropriate plasmids and incubated for 24 h in the dark at 258C before analysis.…”

“…A GFP filter (excitation 470 6 20 nm and emission 525 6 25 nm) was used to detect GFP. For vacuolar staining, protoplasts were resuspended in electroporation buffer (Lukaszewicz et al, 1998) containing 1 mg/mL of Neutral Red (Molecular Probes, Eugene, OR) and incubated for 30 min at room temperature, and then stained protoplasts were observed under visible light. Confocal microscopy was performed using a Bio-Rad MRC-1024 laser scanning system as described previously (Duby et al, 2001).…”