Ana Barbas

The exosome contains domains with specific endoribonuclease, exoribonuclease and cytoplasmic mRNA decay activities

The eukaryotic exosome is a ten-subunit 3′ exoribonucleolytic complex responsible for many RNA-processing and RNA-degradation reactions. How the exosome accomplishes this is unknown. Rrp44 (also known as Dis3), a member of the RNase II family of enzymes, is the catalytic subunit of the exosome. We show that the PIN domain of Rrp44 has endoribonucleolytic activity. The PIN domain is preferentially active toward RNA with a 5′ phosphate, suggesting coordination of 5′ and 3′ processing. We also show that the endonuclease activity is important in vivo. Furthermore, the essential exosome subunit Csl4 does not contain any domains that are required for viability, but its zinc-ribbon domain is required for exosome-mediated mRNA decay. These results suggest that specific exosome domains contribute to specific functions, and that different RNAs probably interact with the exosome differently. The combination of an endoRNase and an exoRNase activity seems to be a widespread feature of RNA-degrading machines.

Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex

RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 Å resolution) and RNA-bound (at 2.74 Å resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented αβ-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the ‘anchor’ and the ‘catalytic’ regions, which act synergistically to provide catalysis. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.

New insights into the mechanism of RNA degradation by ribonuclease II: identification of the residue responsible for setting the RNase II end-product

RNase II is a key exoribonuclease involved in the maturation, turnover, and quality control of RNA. RNase II homologues are components of the exosome, a complex of exoribonucleases. The structure of RNase II unraveled crucial aspects of the mechanism of RNA degradation. Here we show that mutations in highly conserved residues at the active site affect the activity of the enzyme. Moreover, we have identified the residue that is responsible for setting the end product of RNase II. In addition, we present for the first time the models of two members of the RNase II family, RNase R from Escherichia coli and human Rrp44, also called Dis3. Our findings improve the present model for RNA degradation by the RNase II family of enzymes.

Determination of key residues for catalysis and RNA-cleavage specificity: one mutation turns RNase II into a "super"-enzyme

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 single- or 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.”

RNase II: The finer details of the Modus operandi of a molecular killer

The RNase II family of enzymes is ubiquitous and has crucial roles in the processing, degradation and quality control of all types of RNA. These exoribonucleases processively degrade RNA from the 3’-end releasing 5’-nucleotide monophosphates. In prokaryotes, RNase II and RNase R have two N-terminal CSD and one C-terminal S1 domain involved in RNA binding, and a central catalytic RNB domain. In eukaryotes, Rrp44p/Dis3, is a RNase II-like protein with similar modular organization, that is the only catalytically active nuclease in the exosome, a complex crucial for RNA metabolism. Here we review recent progresses in the understanding of the degradation mechanism of RNase II, based on mutational analysis and their characterization regarding catalysis and RNA affinity. We have given particular emphasis on E. coli RNase II but the synergies between the functional and structural studies have shown that our findings have implications in the understanding the similar mode of action of other RNase II family members.

Swapping the domains of exoribonucleases RNase II and RNase R: Conferring upon RNase II the ability to degrade ds RNA

RNase II and RNase R are the two E. coli exoribonucleases that belong to the RNase II super family of enzymes. They degrade RNA hydrolytically in the 3′ to 5′ direction in a processive and sequence independent manner. However, while RNase R is capable of degrading structured RNAs, the RNase II activity is impaired by dsRNAs. The final end‐product of these two enzymes is also different, being 4 nt for RNase II and 2 nt for RNase R. RNase II and RNase R share structural properties, including 60% of amino acid sequence similarity and have a similar modular domain organization: two N‐terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C‐terminal S1 domain. We have constructed hybrid proteins by swapping the domains between RNase II and RNase R to determine which are the responsible for the differences observed between RNase R and RNase II. The results obtained show that the S1 and RNB domains from RNase R in an RNase II context allow the degradation of double‐stranded substrates and the appearance of the 2 nt long end‐product. Moreover, the degradation of structured RNAs becomes tail‐independent when the RNB domain from RNase R is no longer associated with the RNA binding domains (CSD and S1) of the genuine protein. Finally, we show that the RNase R C‐terminal Lysine‐rich region is involved in the degradation of double‐stranded substrates in an RNase II context, probably by unwinding the substrate before it enters into the catalytic cavity. Proteins 2011;

Characterizing Ribonucleases In Vitro : Examples of Synergies between Biochemical and Structural Analysis

The contribution of RNA degradation to the posttranscriptional control of gene expression confers on it a fundamental role in all biological processes. Ribonucleases (RNases) are essential enzymes that process and degrade RNA and constitute one of the main groups of factors that determine RNA levels in the cells. RNase II is a ubiquitous, highly processive hydrolytic exoribonuclease that plays an important role in RNA metabolism. This ribonuclease can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. In this chapter, we explain the general procedures normally used for the characterization of ribonucleases, using as an example a study performed with Escherichia coli RNase II. We present the overexpression and purification of RNase II recombinant enzyme and of a large set of RNase II truncations. We also describe several methods that can be used for biochemically characterizing the exoribonucleolytic activity and studying RNA binding in vitro. Dissociation constants were determined by electrophoretic mobility shift assay (EMSA), surface plasmon resonance (SPR), and filter binding assays using different single- or double-stranded RNA substrates. We discuss the synergies among the biochemical analyses and the structural studies. These methods will be very useful for the study of other ribonucleases.

Comparison of EMSA and SPR for the Characterization of RNA–RNase II Complexes

RNases are enzymes that process and degrade RNA molecules. As such, the study of the interactions between these enzymes and RNA molecules is essential in order to better understand their mechanism of action. In this report, our aim was to use E. coli RNase II as a model to compare two different techniques for the characterization and interpretation of the stability of RNA–protein complexes: Surface Plasmon Resonance and Electrophoretic Mobility Shift Assay.

Modulating the RNA Processing and Decay by the Exosome: Altering Rrp44/Dis3 Activity and End-Product

In eukaryotes, the exosome plays a central role in RNA maturation, turnover, and quality control. In Saccharomyces cerevisiae, the core exosome is composed of nine catalytically inactive subunits constituting a ring structure and the active nuclease Rrp44, also known as Dis3. Rrp44 is a member of the ribonuclease II superfamily of exoribonucleases which include RNase R, Dis3L1 and Dis3L2. In this work we have functionally characterized three residues located in the highly conserved RNB catalytic domain of Rrp44: Y595, Q892 and G895. To address their precise role in Rrp44 activity, we have constructed Rrp44 mutants and compared their activity to the wild-type Rrp44. When we mutated residue Q892 and tested its activity in vitro, the enzyme became slightly more active. We also showed that when we mutated Y595, the final degradation product of Rrp44 changed from 4 to 5 nucleotides. This result confirms that this residue is responsible for the stacking of the RNA substrate in the catalytic cavity, as was predicted from the structure of Rrp44. Furthermore, we also show that a strain with a mutation in this residue has a growth defect and affects RNA processing and degradation. These results lead us to hypothesize that this residue has an important biological role. Molecular dynamics modeling of these Rrp44 mutants and the wild-type enzyme showed changes that extended beyond the mutated residues and helped to explain these results.

Antigenic response to CT-P13 and infliximab originator in inflammatory bowel disease patients shows similar epitope recognition

To test the cross‐immunogenicity of anti‐CT‐P13 IBD patients’ sera to CT‐P13/infliximab originator and the comparative antigenicity evoked by CT‐P13/infliximab originator sera.