Poster: Regulation of gene expression

Abs # 786: Functional analysis of AtRNase II, a nucleus-encoded chloroplast exoribonuclease

Presenter:	Gutierrez, Ryan , rg224@cornell.edu
Authors	Gutierrez, Ryan  (A)   Bollenbach, Thomas J. (A)   Stern, David B. (A)
Affiliations:	(A): Boyce Thompson Institute for Plant Research, Cornell University

Synthesis of thylakoid proteins, and therefore photosynthesis, relies on the proper accumulation and processing of transcripts encoded by the chloroplast genome. The major mechanism controlling RNA accumulation is at the level of RNA stability, where nucleus-encoded ribonucleases mediate turnover. Degradation of chloroplast transcripts begins with endonucleolytic cleavage and is followed by polyadenylation and 3’-5’ exonucleolytic degradation. In E. coli, the RNR gene family encodes 3’-5’ exonucleases that participate in RNA processing and in turnover of polyadenylated transcripts. We therefore searched the Arabidopsis genome for RNase II/RNase R homologues and found two putative chloroplast-targeted ribonucleases, which we have called AtRNase II and AtRNase R. Using RNase II-YFP fusion proteins and transient expression assays, we have verified that this enzyme is chloroplast-targeted. We have obtained three T-DNA mutant lines for RNase II from the SIGnAL collection at the Salk Institute. Homozygous mutant seedlings have white cotyledons and pale leaves and grow more slowly than WT plants. RNA filter blots show that while accumulation of psbA and rbcL transcripts is unaffected by the mutation, homozygous mutants exhibit significant decreases in the accumulation of fully processed 16S, 23S, 4.5S and 5S ribosomal RNAs. These defects in rRNA processing suggest that fewer ribosomes are present in the mutant chloroplasts, resulting in a lower rate of protein synthesis, and therefore a lower accumulation of thylakoid proteins. This is being tested by analyzing thylakoid protein accumulation and by measuring the amounts of actively translating ribosomes (via polysome analysis). This research is funded by an ASPB SURF fellowship to RG and a U.S. D.O.E. Biosciences Grant to DS.