Inês Silva

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.

Regulation of the small regulatory RNA MicA by ribonuclease III: a target-dependent pathway

MicA is a trans-encoded small non-coding RNA, which downregulates porin-expression in stationary-phase. In this work, we focus on the role of endoribonucleases III and E on Salmonella typhimurium sRNA MicA regulation. RNase III is shown to regulate MicA in a target-coupled way, while RNase E is responsible for the control of free MicA levels in the cell. We purified both Salmonella enzymes and demonstrated that in vitro RNase III is only active over MicA when in complex with its targets (whether ompA or lamB mRNAs). In vivo, MicA is demonstrated to be cleaved by RNase III in a coupled way with ompA mRNA. On the other hand, RNase E is able to cleave unpaired MicA and does not show a marked dependence on its 5′ phosphorylation state. The main conclusion of this work is the existence of two independent pathways for MicA turnover. Each pathway involves a distinct endoribonuclease, having a different role in the context of the fine-tuned regulation of porin levels. Cleavage of MicA by RNase III in a target-dependent fashion, with the concomitant decay of the mRNA target, strongly resembles the eukaryotic RNAi system, where RNase III-like enzymes play a pivotal role.

The role of RNases in the regulation of small RNAs

Ribonucleases (RNases) are key factors in the control of biological processes, since they modulate the processing, degradation and quality control of RNAs. This review gives many illustrative examples of the role of RNases in the regulation of small RNAs (sRNAs). RNase E and PNPase have been shown to degrade the free pool of sRNAs. RNase E can also be recruited to cleave mRNAs when they are interacting with sRNAs. RNase III cleaves double-stranded structures, and can cut both the sRNA and its RNA target when they are hybridized. Overall, ribonucleases act as conductors in the control of sRNAs. Therefore, it is very important to further understand their role in the post-transcriptional control of gene expression.

SATP (YaaH), a succinate–acetate transporter protein in Escherichia coli

In the present paper we describe a new carboxylic acid transporter in Escherichia coli encoded by the gene yaaH. In contrast to what had been described for other YaaH family members, the E. coli transporter is highly specific for acetic acid (a monocarboxylate) and for succinic acid (a dicarboxylate), with affinity constants at pH 6.0 of 1.24±0.13 mM for acetic acid and 1.18±0.10 mM for succinic acid. In glucose-grown cells the ΔyaaH mutant is compromised for the uptake of both labelled acetic and succinic acids. YaaH, together with ActP, described previously as an acetate transporter, affect the use of acetic acid as sole carbon and energy source. Both genes have to be deleted simultaneously to abolish acetate transport. The uptake of acetate and succinate was restored when yaaH was expressed in trans in ΔyaaH ΔactP cells. We also demonstrate the critical role of YaaH amino acid residues Leu¹³¹ and Ala¹⁶⁴ on the enhanced ability to transport lactate. Owing to its functional role in acetate and succinate uptake we propose its assignment as SatP: the Succinate-Acetate Transporter Protein.

Prevalence of rheumatic and musculoskeletal diseases and their impact on health-related quality of life, physical function and mental health in Portugal: results from EpiReumaPt– a national health survey

Objectives To estimate the national prevalence of rheumatic and musculoskeletal diseases (RMDs) in the adult Portuguese population and to determine their impact on health-related quality of life (HRQoL), physical function, anxiety and depression. Methods EpiReumaPt is a national health survey with a three-stage approach. First, 10 661 adult participants were randomly selected. Trained interviewers undertook structured face-to-face questionnaires that included screening for RMDs and assessments of health-related quality of life, physical function, anxiety and depression. Second, positive screenings for ≥1 RMD plus 20% negative screenings were invited to be evaluated by a rheumatologist. Finally, three rheumatologists revised all the information and confirmed the diagnoses according to validated criteria. Estimates were computed as weighted proportions, taking the sampling design into account. Results The disease-specific prevalence rates (and 95% CIs) of RMDs in the adult Portuguese population were: low back pain, 26.4% (23.3% to 29.5%); periarticular disease, 15.8% (13.5% to 18.0%); knee osteoarthritis (OA), 12.4% (11.0% to 13.8%); osteoporosis, 10.2% (9.0% to 11.3%); hand OA, 8.7% (7.5% to 9.9%); hip OA, 2.9% (2.3% to 3.6%); fibromyalgia, 1.7% (1.1% to 2.1%); spondyloarthritis, 1.6% (1.2% to 2.1%); gout, 1.3% (1.0% to 1.6%); rheumatoid arthritis, 0.7% (0.5% to 0.9%); systemic lupus erythaematosus, 0.1% (0.1% to 0.2%) and polymyalgia rheumatica, 0.1% (0.0% to 0.2%). After multivariable adjustment, participants with RMDs had significantly lower EQ5D scores (β=−0.09; p<0.001) and higher HAQ scores (β=0.13; p<0.001) than participants without RMDs. RMDs were also significantly associated with the presence of anxiety symptoms (OR=3.5; p=0.006). Conclusions RMDs are highly prevalent in Portugal and are associated not only with significant physical function and mental health impairment but also with poor HRQoL, leading to more health resource consumption. The EpiReumaPt study emphasises the burden of RMDs in Portugal and the need to increase RMD awareness, being a strong argument to encourage policymakers to increase the amount of resources allocated to the treatment of rheumatic patients.

Adaptable stirred-tank culture strategies for large scale production of multicellular spheroid-based tumor cell models.

Currently there is an effort toward the development of in vitro cancer models more predictive of clinical efficacy. The onset of advanced analytical tools and imaging technologies has increased the utilization of spheroids in the implementation of high throughput approaches in drug discovery. Agitation-based culture systems are commonly proposed as an alternative method for the production of tumor spheroids, despite the scarce experimental evidence found in the literature. In this study, we demonstrate the robustness and reliability of stirred-tank cultures for the scalable generation of 3D cancer models. We developed standardized protocols to a panel of tumor cell lines from different pathologies and attained efficient tumor cell aggregation by tuning hydrodynamic parameters. Large numbers of spheroids were obtained (typically 1000-1500 spheroids/mL) presenting features of native tumors, namely morphology, proliferation and hypoxia gradients, in a cell line-dependent mode. Heterotypic 3D cancer models, based on co-cultures of tumor cells and fibroblasts, were also established in the absence or presence of additional physical support from an alginate matrix, with maintenance of high cell viability. Altogether, we demonstrate that 3D tumor cell model production in stirred-tank culture systems is a robust and versatile approach, providing reproducible tools for drug screening and target verification in pre-clinical oncology research.

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. WIREs RNA 2013, 4:607–615. doi: 10.1002/wrna.1180

Surprises in the 3′‐end: ‘U’ can decide too!

RNA molecules are subjected to post‐transcriptional modifications that might determine their maturation, activity, localization and stability. These alterations can occur within the RNA molecule or at its 5′‐ or 3′‐ extremities, and are essential for gene regulation and proper function of the RNA. One major type of modification is the 3′‐end addition of nontemplated nucleotides. Polyadenylation is the most well studied type of 3′‐RNA modification, both in eukaryotes and prokaryotes. The importance of 3′‐oligouridylation has recently gained attention through the discovery of several types of uridylated‐RNAs, by the existence of enzymes that specifically add poly(U) tails and others that preferentially degrade these tails. Namely, Dis3L2 is a 3′–5′ exoribonuclease from the RNase II/RNB family that has been shown to act preferentially on oligo(U)‐tailed transcripts. Our understanding of this process is still at the beginning, but it is already known to interfere in the regulation of diverse RNA species in most eukaryotes. Now that we are aware of the prevalence of RNA uridylation and the techniques available to globally evaluate the 3′‐terminome, we can expect to make rapid progress in determining the extent of terminal oligouridylation in different RNA populations and unravel its impact on RNA decay mechanisms. Here, we sum up what is known about 3′‐RNA modification in the different cellular compartments of eukaryotic cells, the conserved enzymes that perform this 3′‐end modification and the effectors that are selectively activated by this process.

Recovery of antioxidant and antiproliferative compounds from watercress using pressurized fluid extraction

In this work, pressurized fluid extraction was explored to recover isothiocyanates (ITCs) and phenolic compounds from watercress. Pretreatment of the raw material was studied, using different conditions of temperature (25 °C; 35 °C), pressure (Patm; 25 MPa), incubation time (0; 30; 60; 120 min) and moisture content (125; 250; 900% of water, dry basis), aiming at promoting the enzymatic hydrolysis of glucosinolates in ITCs. Extractions of ITCs were performed with supercritical CO2, and different mixtures of CO2 : ethanol (0–50, % w/w) were applied to obtain phenolic-enriched ITCs extracts. Extractions were performed at 35 °C and 25 MPa for 2 h and extracts analyzed concerning total ITCs, phenethyl isothiocyanate (PEITC) content, phenolic composition, antioxidant capacity (using ORAC, HORAC and HOSC assays) and antiproliferative effect in a human colorectal cancer cell line (HT-29). Results showed that supercritical CO2 was highly selective in isolating ITCs from watercress (up to 31.7 ± 1.6 μmol ITC per g). When mixtures of CO2 : ethanol were used, extraction of phenolics (up to 10.1 ± 0.8 mg GAE per g) and antioxidants (up to 204.4 ± 21.5 μmol TE per g concerning ORAC, 70.8 ± 10.7 μmol CAE per g for HORAC and 189.5 ± 22.9 μmol TE per g for HOSC), was promoted. PEITC was the main compound responsible for the inhibition of cancer cell growth of all extracts (EC5024 h = 27.8 ± 1.9 μM PEITC). However, extracts obtained by supercritical CO2 extraction after a 30 minute incubation period with CO2 and with CO2 : ethanol mixtures (80 : 20 and 60 : 40, % w/w) were revealed to be effective in isolating other bioactive compounds that enhanced the antiproliferative response of extracts (EC5024 h values of 23.1 ± 0.9; 20.7 ± 1.9 and 19.8 ± 0.7 μM PEITC, respectively).

Formulation of pea protein for increased satiety and improved foaming properties

Pea protein has been associated with promoting the satiety effect. One of the issues associated with the incorporation of pea protein in food products is the product homogeneity due to its solubility and dispersibility issues. Within this context, one goal of this study was to exploit the use of supercritical fluid technology to develop Solid Lipid Pea Particle (SLPP) aiming at improving dispersibility in fat-based products. PP was encapsulated by the PGSS® (Particles from Gas Saturated Solutions) technique into glyceryl dipalmitostearate (E471) and olive oil. Different process conditions, namely pressure (7.3–20.7 MPa), temperature (51–75 °C) and equilibrium time (3–37 min) were tested in order to optimize the encapsulation of pea protein via Response Surface Methodology (RSM), following a Central Composite Rotatable Design (CCRD). Results showed that pressure and the interaction between pressure and temperature had a significant impact (p < 0.05) on the protein load and thus on the encapsulation efficiency. The highest encapsulation efficiency (96%) was achieved at 14 MPa, 51 °C and 20 min. Under these conditions, SLPP presented 0.15 mg of protein per mg of particles and 84% of lipase inhibitory activity. When compared with the PP (non-encapsulated), liposoluble pea protein particles contributed to a better product homogenization. The food industry can also take advantage of the ability of pea protein for foam stabilization in aqueous food products. Therefore, PP was treated with high-pressure supercritical CO2 treatment (HPT-scCO2) that has led to improved foaming properties when compared with the non-treated PP.