Expression profiling of second step splicing factors and role of PRP18a, a second step splicing factor in arabidopsis thaliana

Annamalai, Devi

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Description

Title

Expression profiling of second step splicing factors and role of PRP18a, a second step splicing factor in arabidopsis thaliana

Author(s)

Annamalai, Devi

Issue Date

2011-05-25T14:39:40Z

Director of Research (if dissertation) or Advisor (if thesis)

Schuler, Mary A.

Doctoral Committee Chair(s)

Schuler, Mary A.

Committee Member(s)

• Zelinski, Ray
• Chessman, John
• Ming, Ray R.

Department of Study

School of Integrative Biology

Discipline

Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Arabidopsis thaliana
• RNA splicing
• PRP18a
• SLU7-2
• Snu116
• plants
• abiotic stress
• boric acid

Abstract

Pre-mRNA splicing represents one of the most fundamental processes that is performed by snRNAs, proteins and a variety of non-snRNP proteins including PRP16, PRP17, PRP18, PRP22 and SLU7. In contrast to yeast and mammals, which contain single gene for each of their second step splicing factors, Arabidopsis thaliana has small subfamilies for PRP17, PRP18, PRP22, SLU7 and a single gene for PRP16. Results presented here attempt to understand the requirement for multiple versions of these second step splicing factors in plant intron recognition. To determine if various members of each subfamily are functionally redundant or have additional functions, I have also analyzed their expression patterns in different tissues of 4-week-old soil- grown plants and also in response to different chemical stresses in 3-week-old-plate grown seedlings. Molecular analyses have indicated that the second step splicing factor mRNAs are differentially expressed in rosette, shoot and cauline tissues. They also have indicated that many are expressed at substantially higher levels in response to cadmium sulfate and mercuric chloride. In contrast, the second step splicing factor transcripts are not affected by copper and mercuric sulfate. Pre-mRNA transcripts for PRP17-2 and PRP22-2 accumulate with zinc and cadmium sulfate and, to some extent, with mercuric chloride. In contrast, pre-mRNA transcripts for PRP22-3, PRP22-2 and PRP22-1 decrease in copper- and mercuric acetate-treated seedlings. Intriguingly, there was completely different second step splicing factors expression pattern observed in the presence of two different salt forms of mercury (mercuric chloride and mercuric acetate) with mercuric chloride having harsh effects on the expression of second step splicing factors. In contrast to yeast and mammalian systems that contain one locus designated as PRP18, Arabidopsis thaliana contains two loci designated as AtPRP18a and AtPRP18b. The substantially longer PRP18a protein shares 23-40% identity with its homologs in yeast and mammals and includes one highly conserved loop region present in other PRP18 proteins. In addition, it contains a nuclear localization signal (NLS), a putative G-domain for GTP binding and a putative potassium regulatory channel inactivation domain. Analyses of homozygous plants of the SALK_131180 T-DNA knockout line have indicated that this gene is essential for normal growth and established its role in many aspects of plant development and alternative splicing. Phenotypic analyses have indicated that Atprp18a knockout plants exhibit pleiotropic defects. Molecular analyses indicate that complete elimination of AtPRP18a transcripts differentially affects a number of target genes in a variety of tissues. Again, in contrast to yeast and mammalian systems that contain one locus designated as SLU7, Arabidopsis thaliana contains three loci designated as SLU7-1a, SLU7-1b and SLU7-2 and AtSLU7-2 does not appear to compensate for PRP18’s role in plant splicing and, instead, it is downregulated in reproductive tissues of Atprp18a knockout plants. In buds of three knockout plants potential cryptic splice variants of AtNPGR1 (No Pollen Germination-1) transcripts were eliminated and, related AtNPGR2 transcripts. In addition, in Atprp18a knockouts showed variation in the splicing pattern of AtRSZ33 transcripts with cryptic proximal 3’splice sites establishing a role for AtPRP18a in 3’splice site selection. Surprisingly, other second step splicing factors such as PRP16, PRP17-1, PRP17-2, SLU7-1a and SLU7-1b were not affected in Atprp18a knockout plants. Comparisons of the introns present in some of the transcripts suggest that PRP18a exerts its pleiotropic role in plant development because it is needed for recognition of a subset of introns having weak 5’ and 3’ splice site consensus shown in other eukaryotic systems to interact with U5-snRNA loop I. Boron is an essential micronutrient for plant growth with the important roles in nucleic acid metabolism, cell wall synthesis, cell wall structure, carbohydrate and protein metabolism, indole acetic acid metabolism, membrane integrity and function, and phenol metabolism in plants and animals. Even so, its molecular role is still not determined in all of these processes. Recently, it has been shown that excess of boric acid induces overexpression of genes involved in RNA splicing, mRNA degradation, nuclear export of RNA, transcription and translation are overexpressed (AtPAB2, AtRBP47c, AtRPS20B, AtMYB13 and AtMYB68) potentially connecting the presence of boric acid to the effects on RNA-dependent processes in Arabidopsis thaliana. To define boron’s effect on second step of splicing factors in Arabidopsis, where multiple loci coding for almost every second step splicing factors, the effects of 1mM and 5mM boric acid on Atprp18a homozygous knockout plants. These analyses demonstrated that SLU7-2 transcripts are downregulated and AtU5-Snu114-3 transcripts are upregulated in 5 mM boric acid. In addition, 1 mM boric acid-treated Atprp18a knockout seedlings, showed increased accumulation of NPGR2 transcripts.

Graduation Semester

2011-05

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Copyright 2011 Devi Annamalai

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