Effects of the multiple polyadenylation signal AAUAAA on mRNA 3′-end formation and gene expression

Abstract

Polyadenylation (poly(A)) of eukaryotic mRNA is a critical step for gene expression. In plants, poly(A) signals leading to the formation of polyadenosine tails after mRNAs include the far upstream elements, the AAUAAA-like signals, and the mRNA cleavage sites for poly(A). Multiple AAUAAA signals leading to alternative polyadenosine formation have been found in many genes, but the effects of each AAUAAA signal on gene expression remain to be uncovered. A DNA fragment, whose transcript contains two canonical AAUAAA signals from the 3'-untranslation region of endochitinase gene of tobacco (Nicotiana tabacum L. cv. W38), was mutated and constructed into the downstream of beta-glucuronidase (GUS) coding region. Transient expression of GUS gene from these constructs indicated that the distal AAUAAA signal from the stop codon was more important than the proximal one in stimulating gene expression. Also, the sequence rather than the distance between the stop codon and the AAUAAA signal region was critical for gene expression. Transgenic tobaccos with these constructs were also generated, and the position of the polyadenosine tail formation in this region was mapped. Results revealed that both AAUAAA signals were functional, and that polyadenosine tails of most transcripts were directed by the distal AAUAAA signal. Finally, the RNA stabilities of these variants in transgenic plants were measured. RNAs from the variants with the functional distal AAUAAA signal were more stable than those with the functional proximal one only. The possible secondary structure in this poly(A) signal region was predicted and discussed.

Recombinant expression of LFchimera antimicrobial peptide in a plant-based expression system and its antimicrobial activity against clinical and phytopathogenic bacteria

“…The constitutive cauliflower mosaic virus 35S (CaMV35S) promoters are most frequently utilized to enhance the expression of recombinant proteins in dicotyledons such as tobacco [48,56,57]. Polyadenylation sites downstream of the stop codon are critical for RNA processing; they also influence the stability of the transcript and the level of protein expression [58]. Widely used polyadenylation sites include those of the CaMV 35S transcript and the Agrobacterium NOS transcript [57,59].…”

Successful use of Nicotiana tabacum hairy roots for the recombinant production of Cecropin A peptide

“…1). Polyadenylation is necessary for RNA processing, the stability of transcripts, and the level of protein expression 19 . For increasing the translation efficiency and improving the recombinant protein expression, codon optimization is very necessary 20 .…”

“…Polyadenylation is necessary for RNA processing, the stability of transcripts, and the level of protein expression. 19 For increasing the translation efficiency and improving the recombinant protein expression, codon optimization is very necessary. 20 The Cecropin A is a peptide with an animal origin; therefore, its codons were optimized based on the codon usage of tobacco.…”

Alternative polyadenylation and gene expression regulation in plants

“…Such a result could be viewed as two overlapping 6‐nt signals defining the same poly(A) site. A recent report found that the use of two AAUAAA signals in the same 3′‐UTR, but with different surrounding sequences, may result in differential expression levels of transcripts,38 which indicates that the strength of the signals can be modulated by adjacent signals such as FUE and CE (see below).…”