Transcriptional and post-transcriptional enhancement of gene expression by the 5′ UTR intron of rice rubi3 gene in transgenic rice cells

Abstract

Introns play a very important role in regulating gene expression in eukaryotes. In plants, many introns enhance gene expression, and the effect of intron-mediated enhancement (IME) of gene expression is reportedly often more profound in monocots than in dicots. To further gain insight of IME in monocot plants, we quantitatively dissected the effect of the 5' UTR intron of the rice rubi3 gene at various gene expression levels in stably transformed suspension cell lines. The intron enhanced the GUS reporter gene activity in these lines by about 29-fold. Nuclear run-on experiments demonstrated a nearly twofold enhancement by the 5' UTR intron at the transcriptional level. RNA analysis by RealTime quantitative RT-PCR assays indicated the intron enhanced the steady state RNA level of the GUS reporter gene by nearly 20-fold, implying a strong role of the intron in RNA processing and/or export. The results also implicated a moderate role of the intron in enhancement at the translational level ( approximately 45%). Moreover, results from a transient assay experiment using a shortened exon 1 sequence revealed an important role of exon 1 of rubi3 in gene expression. It may also hint a divergence in IME mechanisms between plant and animal cells. These results demonstrated transcriptional enhancement by a plant intron, but suggested that post-transcriptional event(s) be the major source of IME.

Two novel positive cis-regulatory elements involved in green tissue-specific promoter activity in rice (Oryza sativa L ssp.)

“…The oil palm metallothionein promoter (MSP1 promoter) is a fruit-specific expression promoter, deletion analysis revealed that 5 0 -UTR served as a strong positive regulatory region for mesocarp-specific expression, but it had no influence on the level of expression in response to abiotic stresses (Omidvar et al 2010). The 5 0 UTR intron of the rice rubi3 gene enhanced the GUS reporter gene activity in stable transformed suspension cell lines by about 29-fold, and RNA analysis by qRT-PCR assays indicated that this region enhanced the steady state RNA level of the GUS reporter gene by nearly 20-fold (Samadder et al 2008). GSE2 (located at ?1 to ?16) is a cis-acting element and locates within 5 0 -UTR region of P DX1 , also functions as a positive regulator in green tissues (sheath and stem), although the regulation effect is not very strong when comparing with GSE1.…”

Isolation and Characterization of a Polyubiquitin Gene and Its Promoter Region fromMesembryanthemum crystallinum

“…43) On the other hand, in maize Sh1 gene intron 1, intron splicing was essential for IME, as shown by the fact that mutations at the 5 0 and 3 0 splice sites of the first intron blocked transcript splicing and reduced promoter activity in transient assays by 2-fold. 44) Recently, after analyzing the leader intron of the rice rubi3 gene, posttranscriptional events were suggested to be the major source of IME, 45) although Rose 46) reported that IME is mostly a transcriptional event in plants. Thus, enhancement of the activities of promoters by a leader intron is still unclear and there might be some fundamental differences in the mechanism of IME between monocots and dicots.…”

Strategies to Increase Heterologous Protein Expression in Rice Grains

“…The poly (A) tail, in particular, plays an important role in determining transcript stability and function (Hunt 2008;Samadder et al 2008), and a poor 3′′UTR can greatly reduce transcript stability (Green 1993). Incorporating 3′UTRs harboring these elements can increase gene expression (Dong et al 2007).…”