RNAs are extremely important molecules inside the cell, which perform many different functions. For example, messenger RNAs, transfer RNAs and ribosomal RNAs are involved in protein synthesis, whereas noncoding RNAs have numerous regulatory roles. Ribonucleases (RNases) are the enzymes responsible for the processing and degradation of all types of RNAs, having multiple roles in every aspect of RNA metabolism. However, the involvement of RNases in disease is still not well understood. This review focuses on the involvement of the RNase II/RNB family of 3'-5' exoribonucleases in human disease. This can be attributed to direct effects, whereby mutations in the eukaryotic enzymes of this family [defective in sister chromatid joining (Dis3; or Rrp44), Dis3-like exonuclease 1 (Dis3L1; or Dis3L) and Dis3-like exonuclease 2 (Dis3L2)] are associated with a disease, or indirect effects, whereby mutations in the prokaryotic counterparts of RNase II/RNB family (RNase II and/or RNase R) affect the physiology and virulence of several human pathogens. In this review, we compare the structural and biochemical characteristics of the members of the RNase II/RNB family of enzymes. The outcomes of mutations impacting enzymatic function are revisited, in terms of both the direct and indirect effects on disease. Furthermore, we also describe the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exoribonuclease and its importance to combat the COVID-19 pandemic. As a result, RNases may be a good therapeutic target to reduce bacterial and viral pathogenicity. These are the two perspectives on RNase II/RNB family enzymes that are presented in this review.

The transition between exponential and stationary phase is a natural phenomenon for all bacteria and requires a massive readjustment of the bacterial transcriptome. Exoribonucleases are key enzymes in the transition between the two growth phases. PNPase, RNase R and RNase II are the major degradative exoribonucleases in Escherichia coli. We analysed the whole transcriptome of exponential and stationary phases from the WT and mutants lacking these exoribonucleases (Δpnp, Δrnr, Δrnb, and ΔrnbΔrnr). When comparing the cells from exponential phase with the cells from stationary phase more than 1000 transcripts were differentially expressed, but only 491 core transcripts were common to all strains. There were some differences in the number and transcripts affected depending on the strain, suggesting that exoribonucleases influence the transition between these two growth phases differently. Interestingly, we found that the double mutant RNase II/RNase R is similar to the RNase R single mutant in exponential phase while in stationary phase it seems to be closer to the RNase II single mutant. This is the first global transcriptomic work comparing the roles of exoribonucleases in the transition between exponential and stationary phase.

Differential RNA-Seq is a next-generation technology method to determine the significant transcriptomic differences between two and more samples. With this method it is possible to analyze the total RNA content of different samples making it the best global analysis method currently available to study the roles of exoribonucleases in the cell. These enzymes are responsible for the RNA processing and degradation in the cells and therefore affect the total RNA pool in ways not yet fully understood. In Escherichia coli there are three main degradative exoribonucleases RNase II, RNase R, and PNPase that degrade the RNA from the 3' to the 5'-end. These enzymes have several roles in the cell and even though they are degradative enzymes RNase II and PNPase can also protect some RNAs from degradation and PNPase can also act as an RNA polymerase under some conditions. The multiplicity of roles of these exoribonucleases leads to a very high number of transcripts that are affected by their absence in the cell. With the differential RNA-Seq it is possible to obtain a much deeper understanding of how these enzymes work and regulate the bacterial gene expression. In this chapter we have described a differential RNA-Seq data analysis protocol applied to the study of exoribonucleases. We also included the protocol for experimental validation of the RNA-Seq data using qPCR and motility assays. Although the methods described in this chapter were applied to the study of the exoribonucleases, they can also be used for other differential RNA-Seq studies.

The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome.

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.