title: Modulating the RNA processing and decay by the exosome: altering Rrp44/Dis3 activity and end-product.
abstract: 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.
authors.affiliation: Instituto de Tecnologia Química e Biológica - ITQB, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: Filipa P
authors.initials: FP
authors.lastname: Reis
authors.forename: Ana
authors.initials: A
authors.lastname: Barbas
authors.forename: A A
authors.initials: AA
authors.lastname: Klauer-King
authors.forename: Borislava
authors.initials: B
authors.lastname: Tsanova
authors.forename: Daneen
authors.initials: D
authors.lastname: Schaeffer
authors.forename: Eduardo
authors.initials: E
authors.lastname: López-Viñas
authors.forename: Paulino
authors.initials: P
authors.lastname: Gómez-Puertas
authors.forename: Ambro
authors.initials: A
authors.lastname: van Hoof
authors.forename: Cecília M
authors.initials: CM
authors.lastname: Arraiano
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D003062:Codon; D004789:Enzyme Activation; D063326:Exosome Multienzyme Ribonuclease Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055354:Exosomes / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D040081:Protein Array Analysis; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020871:RNA Stability; D012331:RNA, Fungal; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D003062:Codon; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D063326:Exosome Multienzyme Ribonuclease Complex; C542283:DIS3 protein, S cerevisiae