Organizers: Eric Lai (Memorial Sloan Kettering Cancer Center), Benjamin R. tenOever (Mount Sinai School of Medicine), Thomas Tuschl (The Rockefeller University), Jennifer Henry (The New York Academy of Sciences), and Marta Murcia (The New York Academy of Sciences)Presented by the Non-coding RNA Discussion Group
Reported by Kristen Delevich | Posted March 26, 2012
Nucleic acid cleavage is an essential reaction for a wide array of cellular processes. Whether maintaining quality control of cellular mRNA or generating non-coding RNAs, nucleases are central to proper cell function. Speakers attending The Cellular Functions of RNA Nucleases symposium, presented by the Non-coding RNA Discussion Group on November 2, 2011, discussed the diverse roles that cellular nucleases perform in the biogenesis or quality control of rRNAs, mRNAs, and miRNAs. The forum focused on unique and common structural features among different nucleases and the diseases associated with the nucleases' malfunction.
Stefanie Gerstberger, a graduate student at the Rockefeller University, started the meeting with an introduction to the conserved function and expression profiles of RNA nucleases. RNA nucleases are enzymes that cleave the phosphodiester bonds that join nucleotides along the backbones of RNA polymer chains. The simplest distinction to draw among the RNA nucleases is where along a polynucleotide chain they cleave: exonucleases work from either the 3′ or 5′ ends, cleaving one nucleotide at a time, while endonucleases cleave within the polynucleotide chain.
Under most conditions the phosphorous-oxygen (P-O) bond is highly stable, but nucleases utilize a variety of nucleophiles to attack one of the two bridging scissile phosphates and to initiate a bimolecular nucleophilic substitution reaction (SN2, for short). The most commonly used nucleophile is a deprotonated water molecule (OH-) that mediates nucleophilic attack and direct hydrolysis of the P-O bond. Although the fundamental chemistry of RNA cleavage is the same, there is a remarkable diversity and complexity in RNA nuclease structure and catalytic mechanisms.
Gerstberger delineated catalytic mechanisms of RNA nucleases along the lines of their dependence on metal ions. Nucleases that use external nucleophiles (like deprotonated water) are metal-dependent because metal ions activate a nucleophile and stabilize the transition state of the ensuing bimolecular nucleophilic substitution reaction. On the other hand, metal-independent RNA nucleases use the 2′-hydroxyl (OH-) of ribose internal to the RNA structure itself as a nucleophile. Metal-independent RNA nucleases invariably generate a 2′,3′ cyclic phosphate intermediate. Generally speaking, metal-independent nucleases exhibit less substrate specificity than their metal-dependent counterparts and require greater regulation. This means that metal-independent nucleases are more often spatially restricted and kept in check by specific inhibitors. All known exonucleases are metal-dependent, while both metal-dependent and metal-independent catalytic mechanisms are found in the endonuclease family.
Gerstberger discussed how the activity of RNA nucleases is required for a diverse set of cellular processes, including maturation and processing of RNA in the nucleus, RNA turnover, degradation of foreign RNA, and regulation of RNA species during stress. The ability of nucleases to specifically recognize their RNA targets is essential to the integrity of these processes and, therefore, to cell survival. It isn't difficult to imagine the disastrous effects of a nuclease run amok, carrying out unwanted or uncontrolled degradation of RNA.
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Jayakrishna Ambati, MD (University of Kentucky)
Susan J. Baserga, MD, PhD (Yale University)
Lorena S. Beese, PhD (Duke University School of Medicine)
Robert Blelloch, MD, PhD (University of California, San Francisco)
Stefanie Gerstberger (The Rockefeller University)
Fedor V. Karginov, PhD (Cold Spring Harbor Laboratory)
Eric Lai, PhD (Memorial Sloan Kettering Cancer Center)
Christopher D. Lima, PhD (Memorial Sloan Kettering Cancer Center)
Benjamin R. tenOever, PhD (Mount Sinai School of Medicine)
Liang Tong, PhD (Columbia University)