The sequence and structure of the 3′-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability

Abstract

A general characteristic of the 3′-untranslated regions (3′ UTRs) of plastid mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops are RNA 3′-end processing signals and determinants of mRNA stability, not transcription terminators. Incubation of synthetic RNAs corresponding to the 3′ UTRs of Chlamydomonas chloroplast genes atpB and petD with a chloroplast protein extract resulted in the accumulation of stable processing products. Synthetic RNAs of the petA 3′ UTR and the antisense strand of atpB 3′ UTR were degraded in the extract. To examine 3′ UTR function in vivo, the atpB 3′ UTR was replaced with the 3′ UTR sequences of the Chlamydomonas chloroplast genes petD, petD plus trnR, rbcL, petA and E. coli thrA by biolistic transformation of Chlamydomonas chloroplasts. Each 3′ UTR was inserted in both the sense and antisense orientations. The accumulation of both total atpB mRNA and ATPase β-subunit protein in all transformants was increased compared to a strain in which the atpB 3′ UTR had been deleted. However, the level of discrete atpB transcripts in transformants containing the antisense 3′ UTR sequences was reduced to approximately one-half that of transformants containing the 3′ UTRs in the sense orientation. These results imply that both the nucleotide sequences and the stem-loop structures of the 3′ UTRs are important for transcript 3′-end processing, and for accumulation of the mature mRNAs.