Studies on endoribonuclease RNase E and its role in RNA turnover

Abstract

RNA turnover is an important level of regulation of gene expression, by which a cell can rapidly adjust to changes in its environment. The turnover of RNA is done by an efficient but not well understood mechanism. The endoribonuclease RNase E in Escherichia coli is an enzyme partitioning in rRNA processing and has been implicated in initiation of mRNA decay.

This thesis provides evidence that E. coli RNase E is functionally interacting with a heat shock protein, GroEL, belonging to the cpn60 family. GroEL was found to co-purify and functionally interact with RNase E in E. coli, and to alter the specificity of the enzyme. This interaction was shown by; immunoprecipitation of RNase E activity with GroEL antibodies, processing of 9S RNAby purified GroEL in vitro and a reduced processing of 9S RNA in a E. coli GroELts-mutant at anon-permissive temperature in vivo. GroEL was also found to bind RNA and to participate in aRNA-binding complex which protects RNA from degradation. The RNA-binding activity is high during slow, anaerobic growth, where also the levels of a modified form of GroEL was found to below.

Three RNA-binding activities could be separated by ion exchange chromatography; in addition to the GroEL-RNA-binding activity, a RNase E containing RNA-binding complex was found. GroEL binds preferentially to ompA 5'-UTR than to the more structured 9S RNA. Human cell extract contain a RNase that resemble RNase E in structure and function. The human enzyme cross-reacts with polyclonal antibodies recognising E. coli RNase E. The RNase E substrates ompA mRNA and 9S RNA are processed in an identical manner by the human and the bacterial RNase E-like enzymes. The human RNase E-like activity cleaved the 3'-untranslated region of c-myc mRNA and RNAs containing repeats of the AUUUA-motifs, implicated in mammalian mRNA degradation, in a similar way as the bacterial RNase E. The half-life of AUUUA-containing RNA is dependent on the number of AUUUA-motifs (in vitro). Halophilic Archaea also contain a RNase E-like activity. Halophilic protein extract processes ompA mRNA and 9S RNA in the same manner as bacterial and eukaryal extracts. Monoclonal antibodies raised against E. coli RNase E recognise polypeptides which could be proteolytic fragments of a archaeal RNase E. The halophilic RNase E-like enzyme is not stable during storage in low salt, while the bacterial and mammalian RNase E-like enzymes do not work under high salt conditions.

These finding indicates that there are RNase E-like activities in all three domains of life. These enzymes recognise the same tested substrates although these substrates do not exist in all domains. Immunological studies show that these enzymes could be related and evolutionary conserved.