Browsing by Author "Sarmah, Bhaskarjyoti"

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Plant pre-mRNA splicing in fission yeast, Schizosaccharomyces pombe
(Academic Press, 2002) Sarmah, Bhaskarjyoti; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis
Pre-mRNA splicing or the removal of introns from precursor messenger RNAs depends on the accurate recognition of intron sequences by the splicing machinery. We have analyzed various aspects of intron sequence and structure in relation to splice site selection and splicing efficiency of a plant gene AmA1 in Schizosaccharomyces pombe. Earlier, we reported the cloning of AmA1, a seed albumin gene from Amaranthus hypochondriacus [A. Raina, A. Datta, Proc. Natl. Acad. Sci. USA 89 (1992) 11774]. In the absence of an in vitro splicing system for plants, the expression of AmA1 genomic clone in S. pombe has been used to analyze splicing of intron constructs. We aim to focus on S. pombe as a possible alternative and examined its effectiveness as a host for plant gene splicing. The results show here that pre-mRNA transcripts of AmA1 gene underwent splicing in S. pombe. Ó 2002 Elsevier Science (USA). All rights reserved.
Premature termination of RNA polymerase II mediated transcription of a seed protein gene in Schizosaccharomyces pombe
(Oxford University Press, 2002) Chakraborty, Subhra; Sarmah, Bhaskarjyoti; Chakraborty, Niranjan; Datta, Asis
The poly(A) signal and downstream elements with transcriptional pausing activity play an important role in termination of RNA polymerase II transcription. We show that an intronic sequence derived from the plant seed protein gene (AmA1 ) specifically acts as a transcriptional terminator in the fission yeast, Schizosaccharomyces pombe. The 3′-end points of mRNA encoded by the AmA1 gene were mapped at different positions in S.pombe and in native cells of Amaranthus hypochondriacus. Deletion analyses of the AmA1 intronic sequence revealed that multiple elements essential for proper transcriptional termination in S.pombe include two site-determining elements (SDEs) and three downstream sequence elements. RT–PCR analyses detected transcripts up to the second SDE. This is the first report showing that the highly conserved mammalian poly(A) signal, AAUAAA, is also functional in S.pombe. The poly(A) site was determined as Y(A) both in native and heterologous systems but at different positions. Deletion of these cis-elements abolished 3′-end processing in S.pombe and a single point mutation in this motif reduced the activity by 70% while enhancing activity at downstream SDE. These results indicate that the bipartite sequence elements as signals for 3′-end processing in fission yeast act in tandem with other cis-acting elements. A comparison of these elements in the AmA1 intron that function as a transcriptional terminator in fission yeast with that of its native genes showed that both require an AT-rich distal and proximal upstream element. However, these sequences are not identical. Transcription run-on analysis indicates that elongating RNA polymerase II molecules accumulate over these pause signals, maximal at 611–949 nt. Furthermore, we demonstrate that the AmA1 intronic terminator sequence acts in a position-independent manner when placed within another gene.