Determination of Key Residues for Catalysis and RNA Cleavage Specificity

Abstract

RNase II is the prototype of a ubiquitous family of enzymes that are crucial for RNA metabolism. In Escherichia coli this protein is a single-stranded-specific 3-exoribonuclease with a modular organization of four functional domains. In eukaryotes, the RNase II homologue Rrp44 (also known as Dis3) is the catalytic subunit of the exosome, an exoribonuclease complex essential for RNA processing and decay. In this work we have performed a functional characterization of several highly conserved residues located in the RNase II catalytic domain to address their precise role in the RNase II activity. We have constructed a number of RNase II mutants and compared their activity and RNA binding to the wild type using different singleor double-stranded substrates. The results presented in this study substantially improve the RNase II model for RNA degradation. We have identified the residues that are responsible for the discrimination of cleavage of RNA versus DNA. We also show that the Arg-500 residue present in the RNase II active site is crucial for activity but not for RNA binding. The most prominent finding presented is the extraordinary catalysis observed in the E542A mutant that turns RNase II into a "super-enzyme."Exoribonuclease II (RNase II) 5 is one of the major exoribonucleases involved in the degradation of RNA (1). This protein degrades RNA hydrolytically in the 3Ј to 5Ј direction, releasing 5Ј-nucleotide monophosphates in a processive manner. Its activity is sequence independent but sensitive to secondary structures, and poly(A) is the preferential substrate for this enzyme (2-6). In Escherichia coli RNase II is responsible for 90% of the hydrolytic activity in crude extracts (7). Moreover, its expression is differentially regulated at the transcriptional and post-transcriptional levels (8 -10), and the protein can be regulated by environmental conditions (10). The E. coli enzyme is the prototype of a widespread family of exoribonucleases, and RNase II homologues can be found in prokaryotes and eukaryotes (11). In the nucleus and in the cytoplasm of eukaryotic cells, RNase II homologues (Rrp44/Dis3) are part of the exosome, an essential multiprotein complex of exoribonucleases involved in processing, turnover, and quality control of different types of RNAs (12). Rrp44 is the only catalytically active nuclease in the human and yeast core exosome (13,14). Moreover, it was recently demonstrated that apart from the RNB catalytic domain, Rrp44p has a second nuclease domain, the PINc domain, that confers endonucleolytic activity to the protein (15, 16). In prokaryotic cells, apart from RNase II, there is an additional RNase II-like enzyme, RNase R, that is involved in RNA degradation and RNA and protein quality control (17)(18)(19)(20). RNase R has also been shown to be required for virulence (21).The determination of E. coli RNase II structure (22-24) showed that, contrary to the sequence predictions, RNase II consisted of four domains; they are two N-terminal cold shock domains (CSD1 and CSD2) involved in RNA b...

Initial genome sequencing and analysis of multiple myeloma

“…First, 3 of the 4 tumors with mutations exhibited loss of heterozygosity via deletion of the remaining DIS3 allele. Second, two of the mutations have been functionally characterized in yeast and bacteria, where they result in loss of enzymatic activity leading to the accumulation of their RNA targets 15, 16 . Given that a key role of the exosome is the regulation of the available pool of mRNAs available for translation 17 , these results suggest that DIS3 mutations may dysregulate protein translation as an oncogenic mechanism in MM.…”

The RNase R from Campylobacter jejuni Has Unique Features and Is Involved in the First Steps of Infection

“…Cj-RNR Is Able to Cleave DNA Substrates-It is known that RNase II, the prototype of this family of enzymes, is able to bind to DNA molecules; however, it is not able to cleave them because there are specific requirements for a ribose in the second or fourth nucleotides counting from the 3Ј-end (43,44). When RNase R was initially characterized, it was shown that is able to cleave DNA, albeit with a reduced efficiency and using higher protein concentrations (34).…”

Selective 2′-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA

“…First, glutamic acid at position 463 is evolutionarily conserved throughout the RNR enzyme family and interacts with the 2′-hydroxyl of RNA in several family members 26,27 . Mutation of this residue in RNase II from glutamic acid to alanine results in a loss of RNA cleavage specificity 28 . Second, although the residue is not always a serine, a hydrogen bonding interaction between a residue in the substrate channel and the nucleotide base-pairing face at N-4 is also highly conserved.…”