Second-step splicing factors and heavy metal stress management in Arabidopsis thaliana
Pre-mRNA splicing in plants is mechanistically the same as splicing in other eukaryotes, but its mode of intron recognition is unique. Whereas in animals a pyrimidine-tract contributes to the definition of exons, in plants, introns are defined by the boundaries of AU-rich intron and GC-rich exon. Some of these differences may depend on splicing factors mediating recognition of the unusual sequence-dependent transition points and/or factors mediating the first- and second-steps in intron definition and excision. The second transesterification step of splicing involves many protein factors (second-step splicing factors) that have not been previously characterized in Arabidopsis. Among these are numerous genes encoded by multicopy genes in this model plant: PRP16 (1 copy), PRP17 (2 copies), PRP18 (2 copies), PRP22 (3 copies) and SLU7 (3 copies). This work is aimed at defining the structural differences and expression patterns of these multiple second-step splicing factors and their involvements in heavy metal stress response. The first goal of this project was to determine unique characteristics of the multicopy second-step splicing factors and the tissue and developmental stages in which each of these genes is expressed. My studies have indicated that all of these second-step splicing factors genes are expressed in Arabidopsis. While most are constitutively expressed throughout each tissue type and developmental stage, a specialized subset including PRP17-2, PRP18B, PRP22-3 and SLU7-2 are primarily expressed in flower and silique tissue in four-week-old plants and throughout the entire seven-week-old plant. Each of these specialized genes has unique structural features when compared to their homologs, suggesting that they form unique spliceosomal networks. These include variations in sequence identity between each other and their homologs in other organisms. Additionally, homology modeling revealed specific sites in which changes in residues will likely contribute to their interactions with other proteins. For example, residues important for protein-protein interaction in ScPRP17 are unique on AtPRP17-2. Basic surface residues on ScPRP18 that contribute to interaction with ScSLU7 are in slightly different locations on both AtPRP18A and AtPRP18B and fewer in number on AtPRP18B. The second goal of this project was to determine the response of second-step splicing factors to heavy metal stress conditions (HgCl2, Hg(OAc)2, CdSO4, CuSO4 and ZnSO4). My studies have shown that plants subjected to increasing concentrations of Hg(OAc)2 and CdSO4 for three weeks from germination accumulate pre-mRNA transcripts of genes not subject to alternative splicing. This effect is observed for some of the second-step splicing factors and some genes that are involved in other cellular processes like plant defense and transcription regulation. In contrast, pre-mRNA transcripts of genes subject to alternative splicing, such as the Ser/Arg-rich (SR) proteins involved in intron recognition, accumulate varied proportions of alternatively spliced transcripts but not pre-mRNA transcripts. To determine whether translation-dependent nonsense-mediated decay (NMD) was involved in the accumulation of pre-mRNAs, the effects of chemically inactivating translation were examined. My studies have shown that the patterns of second-step splicing factor transcript accumulation observed when NMD is chemically knocked out most resemble the patterns of transcript accumulation when plants are treated with cadmium. The fact that these patterns do not match exactly suggests that while the metals may affect NMD, they do not abolish it in the same manner as chemically abolishing it by halting translation and other effects are independent of NMD. It was also determined that many of the second-step splicing factor expression levels are regulated by NMD under normal conditions and the loss of NMD causes an accumulation of these transcripts.
Arabidopsis, pre-mRNA splicing factors, heavy metal stress, Ser/Arg-rich protein, second-step splicing factors
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign