We are delighted to present the Frontiers in Genetics 'Women in RNA' series of article collections. At present, less than 30% of researchers worldwide are women. Long-standing biases and gender stereotypes are discouraging girls and women away from science-related fields, and STEM research in particular. Science and gender equality are, however, essential to ensure sustainable development as highlighted by UNESCO. In order to change traditional mindsets, gender equality must be promoted, stereotypes defeated, and girls and women should be encouraged to pursue STEM careers. The work presented here highlights the diversity of research performed across the entire breadth of RNA research and presents advances in theory, experiment, and methodology with applications to compelling problems. Please note: to be considered for this collection, the first or last author should be a researcher who identifies as a woman.

University of São Paulo

University of Campania Luigi Vanvitelli

Encouraging girls & women to break free from traditional biases in STEM

. This research topic covers a variety of topics related to the genetic disorder Cystic Fibrosis, canine Visceral leishmaniasis, and Leishmania infection. Highlights include: <ul> <li>Drug discovery efforts have led to the development of modulator therapies for Cystic Fibrosis, however, additional therapeutic options are needed.</li> <li>MicroRNAs (miRs) can modulate mRNA translation and regulate immune responses in dogs with CanL.</li> <li>Long noncoding RNAs (lncRNAs) may account for a novel mechanism by which Leishmania can control macrophages.</li> <li>An experimental workflow allows the transcriptomic investigation of single and pooled OE33 cells.</li> </ul>

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Frontiers in Genetics

Frontiers in Molecular Biosciences

Women in RNA: 2022

Cystic fibrosis (CF) is an autosomal recessive disease impacting ∼100,000 people worldwide. This lethal disorder is caused by mutation of the CFtransmembrane conductance regulator (CFTR) gene, which encodes an ATP-binding cassette-class C protein. More than 2,100 variants have been identified throughout the length of CFTR. These defects confer differing levels of severity in mRNA and/or protein synthesis, folding, gating, and turnover. Drug discovery efforts have resulted in recent development of modulator therapies that improve clinical outcomes for people living with CF. However, a significant portion of the CF population has demonstrated either no response and/or adverse reactions to small molecules. Additional therapeutic options are needed to restore underlying genetic defects for all patients, particularly individuals carrying rare or refractory CFTR variants. Concerted focus has been placed on rescuing variants that encode truncated CFTR protein, which also harbor abnormalities in mRNA synthesis and stability. The current mini-review provides an overview of CFTR mRNA features known to elicit functional consequences on final protein conformation and function, including considerations for RNA-directed therapies under investigation. Alternative exon usage in the 5′-untranslated region, polypyrimidine tracts, and other sequence elements that influence splicing are discussed. Additionally, we describe mechanisms of CFTR mRNA decay and post-transcriptional regulation mediated through interactions with the 3′-untranslated region (e.g. poly-uracil sequences, microRNAs). Contributions of synonymous single nucleotide polymorphisms to CFTR transcript utilization are also examined. Comprehensive understanding of CFTR RNA biology will be imperative for optimizing future therapeutic endeavors intended to address presently untreatable forms of CF.

Cystic fibrosis (CF) is an autosomal recessive disease impacting ∼100,000 people worldwide. This lethal disorder is caused by mutation of the CF transmembrane conductance regulator (CFTR) gene, which encodes an ATP-binding cassette-class C protein. More than 2,100 variants have been identified throughout the length of CFTR. These defects confer differing levels of severity in mRNA and/or protein synthesis, folding, gating, and turnover. Drug discovery efforts have resulted in recent development of modulator therapies that improve clinical outcomes for people living with CF. However, a significant portion of the CF population has demonstrated either no response and/or adverse reactions to small molecules. Additional therapeutic options are needed to restore underlying genetic defects for all patients, particularly individuals carrying rare or refractory CFTR variants. Concerted focus has been placed on rescuing variants that encode truncated CFTR protein, which also harbor abnormalities in mRNA synthesis and stability. The current mini-review provides an overview of CFTR mRNA features known to elicit functional consequences on final protein conformation and function, including considerations for RNA-directed therapies under investigation. Alternative exon usage in the 5′-untranslated region, polypyrimidine tracts, and other sequence elements that influence splicing are discussed. Additionally, we describe mechanisms of CFTR mRNA decay and post-transcriptional regulation mediated through interactions with the 3′-untranslated region (e.g. poly-uracil sequences, microRNAs). Contributions of synonymous single nucleotide polymorphisms to CFTR transcript utilization are also examined. Comprehensive understanding of CFTR RNA biology will be imperative for optimizing future therapeutic endeavors intended to address presently untreatable forms of CF.

Features of CFTR mRNA and implications for therapeutics development

Canine Visceral leishmaniasis (CanL) poses a severe public health threat in several countries. Disease progression depends on the degree of immune response suppression. MicroRNAs (miRs) modulate mRNA translation into proteins and regulate various cellular functions and pathways associated with immune responses. MiR-21 and miR-148a can alter the parasite load and M1 macrophages are the principal cells in dogs’ leishmanicidal activity. A previous study found increased miR-21 and miR-148a in splenic leukocytes (SL) of dogs with CanL using microarray analysis and in silico analysis identified PTEN pathway targets. PTEN is involved in the immune regulation of macrophages. We measured PTEN and the production of reactive oxygen species (ROS) and nitric oxide (NO) before and after transfection SLs of dogs with CanL with mimic and inhibition of miR-21 and miR-148a. PTEN levels increased, NO and ROS decreased in SLs from dogs with CanL. Inhibition of miRNA-21 resulted in PTEN increase; in contrast, PTEN decreased after miR-148a inhibition. Nitrite (NO2) levels increased after transfection with miR-21 inhibitor but were decreased with miR-148a inhibitor. The increase in miR-21 promoted a reduction in ROS and NO levels, but miR-148a inhibition increased NO and reduced ROS. These findings suggest that miR-21 and miR-148a can participate in immune response in CanL, affecting PTEN, NO, and ROS levels.

Canine Visceral leishmaniasis (CanL) poses a severe public health threat in several countries. Disease progression depends on the degree of immune response suppression. MicroRNAs (miRs) modulate mRNA translation into proteins and regulate various cellular functions and pathways associated with immune responses. MiR-21 and miR-148a can alter the parasite load and M1 macrophages are the principal cells in dogs’ leishmanicidal activity. A previous study found increased miR-21 and miR-148a in splenic leukocytes (SL) of dogs with CanL using microarray analysis and in silico analysis identified PTEN pathway targets. PTEN is involved in the immune regulation of macrophages. We measured PTEN and the production of reactive oxygen species (ROS) and nitric oxide (NO) before and after transfection SLs of dogs with CanL with mimic and inhibition of miR-21 and miR-148a. PTEN levels increased, NO and ROS decreased in SLs from dogs with CanL. Inhibition of miRNA-21 resulted in PTEN increase; in contrast, PTEN decreased after miR-148a inhibition. Nitrite (NO2) levels increased after transfection with miR-21 inhibitor but were decreased with miR-148a inhibitor. The increase in miR-21 promoted a reduction in ROS and NO levels, but miR-148a inhibition increased NO and reduced ROS. These findings suggest that miR-21 and miR-148a can participate in immune response in CanL, affecting PTEN, NO, and ROS levels.

MicroRNA-21 and microRNA-148a affects PTEN, NO and ROS in canine leishmaniasis

It is well established that infection with Leishmania alters the host cell’s transcriptome. Since mammalian cells have multiple mechanisms to control gene expression, different molecules, such as noncoding RNAs, can be involved in this process. MicroRNAs have been extensively studied upon Leishmania infection, but whether long noncoding RNAs (lncRNAs) are also altered in macrophages is still unexplored. We performed RNA-seq from THP-1-derived macrophages infected with Leishmania amazonensis (La), L. braziliensis (Lb), and L. infantum (Li), investigating a previously unappreciated fraction of macrophage transcriptome. We found that more than 24% of the total annotated transcripts and 30% of differentially expressed (DE) RNAs in Leishmania-infected macrophage correspond to lncRNAs. LncRNAs and protein coding RNAs with altered expression are similar among macrophages infected with the Leishmania species. Still, some species-specific alterations could occur due to distinct pathophysiology in which Li infection led to a more significant number of exclusively DE RNAs. The most represented classes among DE lncRNAs were intergenic and antisense lncRNAs. We also found enrichment for immune response-related pathways in the DE protein coding RNAs, as well as putative targets of the lncRNAs. We performed a coexpression analysis to explore potential cis regulation of coding and antisense noncoding transcripts. We identified that antisense lncRNAs are similarly regulated as its neighbor protein coding genes, such as the BAALC/BAALC-AS1, BAALC/BAALC-AS2, HIF1A/HIF1A-AS1, HIF1A/HIF1A-AS3 and IRF1/IRF1-AS1 pairs, which can occur as a species-specific modulation. These findings are a novelty in the field because, to date, no study has focused on analyzing lncRNAs in Leishmania-infected macrophage. Our results suggest that lncRNAs may account for a novel mechanism by which Leishmania can control macrophage function. Further research must validate putative lncRNA targets and provide additional prospects in lncRNA function during Leishmania infection.

It is well established that infection with Leishmania alters the host cell’s transcriptome. Since mammalian cells have multiple mechanisms to control gene expression, different molecules, such as noncoding RNAs, can be involved in this process. MicroRNAs have been extensively studied upon Leishmania infection, but whether long noncoding RNAs (lncRNAs) are also altered in macrophages is still unexplored. We performed RNA-seq from THP-1-derived macrophages infected with Leishmania amazonensis (La), L. braziliensis (Lb), and L. infantum (Li), investigating a previously unappreciated fraction of macrophage transcriptome. We found that more than 24% of the total annotated transcripts and 30% of differentially expressed (DE) RNAs in Leishmania-infected macrophage correspond to lncRNAs. LncRNAs and protein coding RNAs with altered expression are similar among macrophages infected with the Leishmania species. Still, some species-specific alterations could occur due to distinct pathophysiology in which Li infection led to a more significant number of exclusively DE RNAs. The most represented classes among DE lncRNAs were intergenic and antisense lncRNAs. We also found enrichment for immune response-related pathways in the DE protein coding RNAs, as well as putative targets of the lncRNAs. We performed a coexpression analysis to explore potential cis regulation of coding and antisense noncoding transcripts. We identified that antisense lncRNAs are similarly regulated as its neighbor protein coding genes, such as the BAALC/BAALC-AS1, BAALC/BAALC-AS2, HIF1A/HIF1A-AS1, HIF1A/HIF1A-AS3 and IRF1/IRF1-AS1 pairs, which can occur as a species-specific modulation. These findings are a novelty in the field because, to date, no study has focused on analyzing lncRNAs in Leishmania-infected macrophage. Our results suggest that lncRNAs may account for a novel mechanism by which Leishmania can control macrophage function. Further research must validate putative lncRNA targets and provide additional prospects in lncRNA function during Leishmania infection.

Comparative transcriptomic analysis of long noncoding RNAs in Leishmania-infected human macrophages

Combining phenotypical and molecular characterization of rare cells is challenging due to their scarcity and difficult handling. In oncology, circulating tumor cells (CTCs) are considered among the most important rare cell populations. Their phenotypic and molecular characterization is necessary to define the molecular mechanisms underlying their metastatic potential. Several approaches that require cell fixation make difficult downstream molecular investigations on RNA. Conversely, the DEPArray technology allows phenotypic analysis and handling of both fixed and unfixed cells, enabling a wider range of applications. Here, we describe an experimental workflow that allows the transcriptomic investigation of single and pooled OE33 cells undergone to DEPArray analysis and recovery. In addition, cells were tested at different conditions (unfixed, CellSearch fixative (CSF)- and ethanol (EtOH)-fixed cells). In a forward-looking perspective, this workflow will pave the way for novel strategies to characterize gene expression profiles of rare cells, both single-cell and low-resolution input.

Combining phenotypical and molecular characterization of rare cells is challenging due to their scarcity and difficult handling. In oncology, circulating tumor cells (CTCs) are considered among the most important rare cell populations. Their phenotypic and molecular characterization is necessary to define the molecular mechanisms underlying their metastatic potential. Several approaches that require cell fixation make difficult downstream molecular investigations on RNA. Conversely, the DEPArray technology allows phenotypic analysis and handling of both fixed and unfixed cells, enabling a wider range of applications. Here, we describe an experimental workflow that allows the transcriptomic investigation of single and pooled OE33 cells undergone to DEPArray analysis and recovery. In addition, cells were tested at different conditions (unfixed, CellSearch fixative (CSF)- and ethanol (EtOH)-fixed cells). In a forward-looking perspective, this workflow will pave the way for novel strategies to characterize gene expression profiles of rare cells, both single-cell and low-resolution input.