An Intron Is Required for Dihydrofolate Reductase Protein Stability

Véronique Noé,Simon Mackenzie,Carlos J Ciudad

doi:10.1074/jbc.m212746200

Abstract

We compared the expression of dihydrofolate reductase minigenes with and without an intron. The levels of protein were significantly higher in the presence of dihydrofolate reductase intron 1. However, mRNA levels in both constructs were comparable. In addition, the RNA transcribed from either construct was correctly polyadenylated and exported to the cytoplasm. The intron-mediated increase in dihydrofolate reductase protein levels was position-independent and was also observed when dihydrofolate reductase intron 1 was replaced by heterologous introns. The translational rate of dihydrofolate reductase protein was increased in transfectants from the intron-containing minigene. In addition, the protein encoded by the intronless construct was unstable and subject to lysosomal degradation, thus showing a shorter half-life than the protein encoded by the intron-containing minigene. We conclude that an intron is required for the translation and stability of dihydrofolate reductase protein.

Highlights

We compared the expression of dihydrofolate reductase minigenes with and without an intron
We used a set of dhfr minigenes containing either dhfr intron 1, aprt intron 3, ␤-globin intron 1, or no intronic sequence, and we analyzed the three steps of RNA processing in stable transfectants from these constructs
We show that (i) dihydrofolate reductase (DHFR) mRNA was expressed, correctly polyadenylated, and efficiently accumulated in the cytoplasm despite the absence of an intron in the corresponding minigene, (ii) the presence of an intron in the minigene was a requirement for the accumulation of high levels of DHFR protein, (iii) the effect of an intron on DHFR protein expression was sequence- and position-independent, and (iv) DHFR protein encoded by the intronless minigene was less stable than its counterpart from the intron-containing minigene

Summary

Introduction

We compared the expression of dihydrofolate reductase minigenes with and without an intron. Minigenes derived from intron-containing genes are usually expressed more efficiently than their intronless counterparts [1, 2, 3] This enhanced expression could be because of the presence of intronic enhancers of transcription, the facilitation of polyadenylation by an upstream intron (4 –9), or a requirement for intron removal for the export of mRNA from the nucleus or its stability in the nucleus (10 –13). Efficient transfection by minigenes for dihydrofolate reductase (DHFR) depends on the presence of an intron (18 –20). This requirement can be met by the inclusion of a single intron in mouse or hamster dhfr minigenes. We found that intron removal can influence a cytoplasmic event that is further downstream than translation: the degradation of newly synthesized protein

Methods

Results

Conclusion