The interaction of PRC2 with RNA or chromatin is mutually antagonistic.

Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain genes in a repressed state during development. PRC2 is primarily associated with CpG islands at repressed genes and also possesses RNA binding activity. However, the RNAs that bind PRC2 in cells, the subunits that mediate these interactions, and the role of RNA in PRC2 recruitment to chromatin all remain unclear. By performing iCLIP for PRC2 in comparison with other RNA binding proteins, we show here that PRC2 binds nascent RNA at essentially all active genes. Although interacting with RNA promiscuously, PRC2 binding is enriched at specific locations within RNAs, primarily exon-intron boundaries and the 3' UTR. Deletion of other PRC2 subunits reveals that SUZ12 is sufficient to establish this RNA binding profile. Contrary to prevailing models, we also demonstrate that the interaction of PRC2 with RNA or chromatin is mutually antagonistic in cells and in vitro. RNA degradation in cells triggers PRC2 recruitment to CpG islands at active genes. Correspondingly, the release of PRC2 from chromatin in cells increases RNA binding. Consistent with this, RNA and nucleosomes compete for PRC2 binding in vitro. We propose that RNA prevents PRC2 recruitment to chromatin at active genes and that mutual antagonism between RNA and chromatin underlies the pattern of PRC2 chromatin association across the genome.

Biochemical characterization of the RNase II family of exoribonucleases from the human pathogens Salmonella typhimurium and Streptococcus pneumoniae.

Maturation, turnover, and quality control of RNA are performed by many different classes of ribonucleases. Escherichia coli RNase II is the prototype of the RNase II family of ribonucleases, a ubiquitous family of hydrolytic, processive 3' --> 5' exonucleases crucial in RNA metabolism. RNase R is a member of this family that is modulated in response to stress and has been implicated in virulence. In this work, RNase II-like proteins were characterized in the human pathogens Salmonella typhimurium and Streptococcus pneumoniae. By sequence analysis, only one member of the RNase II family was identified in S. pneumoniae, while both RNase II and RNase R were found in Sa. typhimurium. These enzymes were cloned, expressed, purified, and characterized with regard to their biochemical features and modular architecture. The specificity of substrates and the final products generated by the enzymes were clearly demonstrated. Sa. typhimurium RNase II and RNase R behaved essentially as their respective E. coli counterparts. We have shown that the only hydrolytic RNase found in S. pneumoniae was able to degrade structured RNAs as is the case with E. coli RNase R. Our results further showed that there are differences with regard to the activity and ability to bind RNA from enzymes belonging to two distinct pneumococcal strains, and this may be related to a single amino acid substitution in the catalytic domain. Since ribonucleases have not been previously characterized in S. pneumoniae or Sa. typhimurium, this work provides an important first step in the understanding of post-transcriptional control in these pathogens.

Identification of temperature-sensitive mutations and characterization of thermolabile RNase II variants.

The RNase II family of ribonucleases is ubiquitous and critical for RNA metabolism. The rnb500 allele has been widely used for over 30 years; however, the underlying genetic changes which result in RNase II thermolabile activity remain unknown. Here, we combine molecular and biophysical studies to carry out an in vivo and in vitro investigation of RNase II mutation(s) that confer the rnb500 phenotype. Our findings indicate that RNase II thermolability is due to the Cys284Tyr mutation within the RNB domain, which abolishes activity by increasing protein kinetic instability at the nonpermissive temperature. These findings have important implications for the design of temperature-sensitive variants of other RNase II enzymes, namely those with yet unknown functions.

The RNase II/RNB family of exoribonucleases: putting the 'Dis' in disease.

Important findings over the last years have shed new light onto the mechanistic details of RNA degradation by members of the RNase II/RNB family of exoribonucleases. Members of this family have been shown to be involved in growth, normal chloroplast biogenesis, mitotic control and cancer. Recently, different publications have linked human orthologs (Dis3 and Dis3L2) to important human diseases. This article describes the structural and biochemical characteristics of members of this family of enzymes, and the physiological implications that relate them with disease.

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.

The critical role of RNA processing and degradation in the control of gene expression.

The continuous degradation and synthesis of prokaryotic mRNAs not only give rise to the metabolic changes that are required as cells grow and divide but also rapid adaptation to new environmental conditions. In bacteria, RNAs can be degraded by mechanisms that act independently, but in parallel, and that target different sites with different efficiencies. The accessibility of sites for degradation depends on several factors, including RNA higher-order structure, protection by translating ribosomes and polyadenylation status. Furthermore, RNA degradation mechanisms have shown to be determinant for the post-transcriptional control of gene expression. RNases mediate the processing, decay and quality control of RNA. RNases can be divided into endonucleases that cleave the RNA internally or exonucleases that cleave the RNA from one of the extremities. Just in Escherichia coli there are >20 different RNases. RNase E is a single-strand-specific endonuclease critical for mRNA decay in E. coli. The enzyme interacts with the exonuclease polynucleotide phosphorylase (PNPase), enolase and RNA helicase B (RhlB) to form the degradosome. However, in Bacillus subtilis, this enzyme is absent, but it has other main endonucleases such as RNase J1 and RNase III. RNase III cleaves double-stranded RNA and family members are involved in RNA interference in eukaryotes. RNase II family members are ubiquitous exonucleases, and in eukaryotes, they can act as the catalytic subunit of the exosome. RNases act in different pathways to execute the maturation of rRNAs and tRNAs, and intervene in the decay of many different mRNAs and small noncoding RNAs. In general, RNases act as a global regulatory network extremely important for the regulation of RNA levels.