The ever-evolving concept of the gene: The use of RNA/Protein experimental techniques to understand genome functions

The completion of the human genome sequence together with advances in sequencing technologies have shifted the paradigm of the genome, as composed of discrete and hereditable coding entities, and have shown the abundance of functional noncoding DNA. This part of the genome, previously dismissed as "junk" DNA, increases proportionally with organismal complexity and contributes to gene regulation beyond the boundaries of known protein-coding genes. Different classes of functionally relevant nonprotein-coding RNAs are transcribed from noncoding DNA sequences. Among them are the long noncoding RNAs (lncRNAs), which are thought to participate in the basal regulation of protein-coding genes at both transcriptional and post-transcriptional levels. Although knowledge of this field is still limited, the ability of lncRNAs to localize in different cellular compartments, to fold into specific secondary structures and to interact with different molecules (RNA or proteins) endows them with multiple regulatory mechanisms. It is becoming evident that lncRNAs may play a crucial role in most biological processes such as the control of development, differentiation and cell growth. This review places the evolution of the concept of the gene in its historical context, from Darwin's hypothetical mechanism of heredity to the post-genomic era. We discuss how the original idea of protein-coding genes as unique determinants of phenotypic traits has been reconsidered in light of the existence of noncoding RNAs. We summarize the technological developments which have been made in the genome-wide identification and study of lncRNAs and emphasize the methodologies that have aided our understanding of the complexity of lncRNA-protein interactions in recent years

Gene Expression Profiling in Cancer

The contribution of modern-day genetics in designing efficient gene expression profiles for cancer is immense. The progress of technology and science in recent years provides the opportunity for discovery and application of new techniques for treating various diseases that affect humanity. Methods for finding and analyzing the profile of gene expression of infected cells give scientists the ability to develop more targeted and effective treatments, especially for diseases such as cancer. The development of gene expression profiling is one of the most important achievements in cancer genetics in our time. It is essentially the driving force behind personalized and precision medicine. This book highlights recent developments, applications, and breakthroughs in the field of gene expression profiling in cancer

MicroRNA-mediated regulatory circuits: outlook and perspectives

MicroRNAs have been found to be necessary for regulating genes implicated in almost all signaling pathways, and consequently their dysfunction influences many diseases, including cancer. Understanding of the complexity of the microRNA-mediated regulatory network has grown in terms of size, connectivity and dynamics with the development of computational and, more recently, experimental high-throughput approaches for microRNA target identification. Newly developed studies on recurrent microRNA-mediated circuits in regulatory networks, also known as network motifs, have substantially contributed to addressing this complexity, and therefore to helping understand the ways by which microRNAs achieve their regulatory role. This review provides a summarizing view of the state-of-the-art, and perspectives of research efforts on microRNA-mediated regulatory motifs. In this review, we discuss the topological properties characterizing different types of circuits, and the regulatory features theoretically enabled by such properties, with a special emphasis on examples of circuits typifying their biological significance in experimentally validated contexts. Finally, we will consider possible future developments, in particular regarding microRNA-mediated circuits involving long non-coding RNAs and epigenetic regulators

Molecular determinants of miRNA target specificity and tissue-specific studies in C. elegans

MicroRNAs (miRNAs) control gene expression by repressing target messenger RNAs. Target identification is thus key to understand the biological implications of a miRNA in physiological or pathological processes, but it has remained the main challenge in the field. Traditionally, we refer to “canonical targets” when the 3’UTR of a gene contains a perfect Watson- Crick match to the 5’ sequence of the miRNA. However, many non-canonical miRNA binding sites have been identified that display seed mismatches, pairing beyond the seed or both. In this work, we aimed at understanding the molecular requirements necessary to induce silencing of a transcript by a specific miRNA in vivo. Using genome editing and physiological reporters, we focused on miRNA sharing the same seed sequence (miRNA families) as they permit to understand the involvement of both seed and non-seed pairing. For such investigation we studied the let-7 family of miRNAs because let-7 is conserved in humans and has been found implicated in several pathologies. We performed our studies in C. elegans because this miRNA family has been well characterized in this nematode, and mutant animals have obvious phenotypes that are easy to score. Our results suggest that target specificity of miRNAs belonging to a family depends on the degree of sequence complementarity between the individual miRNA and the transcript. Particularly, pairing of the 3’ sequence of the miRNA is the main determinant to establish preferential binding to a site. In addition, the seed match has a key role in modulating such specificity, as it allows to discriminate between high and low levels of miRNAs. Hence, target specificity of individual miRNAs is not hardwired, but is modulated by the miRNA abundance. We believe that our findings have a broad impact on miRNA target prediction and validation, especially if we want to invest in miRNA therapeutics. Lastly, we show that studying miRNA/target interactions in physiological settings has the power to unequivocally validate targets and expand our knowledge on the miRNA regulatory potential. In parallel, we succeeded in optimizing a FACS-based protocol to isolate worm cells, which we used to profile cell-type specific small RNAs and tissue-specific transcriptomes at single cell resolution. Given the general lack of methods to obtain primary cells and high quality tissue-specific data in the C. elegans community, such results hold the great potential to expand our knowledge about cell-type specific gene expression

Type 1 Diabetic Heart: Examination of Mitochondrial Structure and MicroRNAs

Cardiac complications such as diabetic cardiomyopathy are the leading cause of morbidity and mortality in patients with diabetes mellitus. Dysfunctional mitochondria, an effect associated with cardiomyopathy, are central in the pathogenesis of type 1 diabetes mellitus. Cardiac mitochondria are comprised of two spatially located mitochondria including mitochondria located beneath the sarcolemma, termed subsarcolemmal mitochondria (SSM) and those located in between myofibrils, termed interfibrillar mitochondria (IFM). Mitochondrial subpopulations have been shown to respond differently to pathological and physiological stimuli as reported in a review published by our laboratory. IFM mitochondria are most impacted in a type 1 diabetic setting. Proteomic alterations in cardiac mitochondria during a diabetic insult reveal impact primarily on IFM on nuclear-encoded mitochondrial proteins, a finding that has been previously reported by our laboratory. Alterations of proteins encoded by the mitochondrial genome have not been observed in our proteomics. Further, regulation of nuclear-encoded proteins by microRNAs (miRNAs) has been previously reported by our laboratory. MiRNAs are 22 nucleotide long post-transcriptional regulators with a 7 nucleotide seeding region specific to complementary sequences in the mRNA. More than 30% of all proteins are regulated by miRNAs and one miRNA has the potential to regulate the expression of multiple proteins. The potential regulation of mitochondrial genome encoded proteins by miRNAs has yet to be investigated in mitochondrial subpopulations during diabetes. Among the altered nuclear encoded proteins in type 1 diabetic IFM is structural protein known as mitofilin. Mitofilin is an inner mitochondrial membrane structural protein, well established for its role in maintaining cristae morphology and structure. It is a central component of the mitochondrial contact site and cristae organizing system (MICOS) complex. Interactions of mitofilin with outer and inner membrane proteins have been reported to be crucial for mitochondrial membrane organization, cristae integrity and inner membrane architecture. Moreover, MICOS has been shown to function in concert with ATP synthase dimers. However, association of mitofilin with ATP synthase subunits is not known. Moreover, literature examining association of mitofilin and regulation of mitochondrial genome by miRNAs in type 1 diabetic insult is sparse. Also, the impact of diabetes mellitus on mitofilin protein interactions, mitochondrial structure and function are currently unclear. It is specifically unknown whether overexpression of mitofilin aids in alleviating complications associated with diabetic cardiomyopathy. The goal of the present studies was to determine novel association of mitofilin and the impact of mitofilin overexpression upon mitochondrial structure and function. Further, regulation of mitochondrial genome by mitochondrial miRNAs (mitomiRs) has been investigated. The overall hypothesis of this application is that alterations of cristae morphology, inner membrane organization and mitochondrial dysfunction observed during type 1 diabetic insult are associated with decrements in mitofilin content as well as translational regulation of mitochondrial encoded proteins due to altered levels of mitochondrial miRNAs (mitomiRs). Type 1 diabetes mellitus was induced in five weeks old mice with multiple low dose injections of streptozotocin (STZ) for five consecutive days. Five weeks post hyperglycemic onset, hearts were excised and mitochondrial subpopulations isolated for further studies. Using a gel based technique, mitochondrial proteins immunoprecipitated with mitofilin were subjected to LC-ESI-MS analysis. Proteins from all electron transport chain complexes, structural proteins and proteins involved in protein import were identified in an immunoprecipitated complex. Association of mitofilin with F0 -ATP synthase subunit b (ATP5F1) was decreased in the diabetic IFM when compared with control. Moreover, interaction of mitofilin with coiled-coil-helix coiled-coil-helix domain 3 (CHCHD3) trended towards decreased in diabetic IFM. A transgenic mouse line overexpressing mitofilin was generated and utilized to investigate the role of mitofilin overexpression in mitochondrial structure and function. Restoration of ejection fraction and fractional shortening was observed in mitofilin diabetic mice as compared to wild-type controls (P\u3c0.05 for both). Decrements observed in electron transport chain (ETC) complexes I, III, IV and V activities, state 3 respiration, lipid peroxidation as well as mitochondria membrane potential in type 1 diabetic IFM were restored in mitofilin diabetic mice (P\u3c0.05 for all). Qualitative analyses of electron micrographs revealed restoration of mitochondrial cristae structure in mitofilin diabetic mice as compared to wild-type controls. Furthermore measurement of mitochondrial internal complexity using flow cytometry displayed significant reduction in internal complexity in diabetic IFM which was restored in mitofilin diabetic IFM (P\u3c0.05). No significant changes in mitochondrial dynamic regulating proteins or mitochondrial DNA content were observed. Examination of mitochondrial miRNAs was performed using microarray technology coupled with cross-linking immunoprecipitation and next generation sequencing, we identified a functional pool of mitochondrial microRNAs, termed mitomiRs that are redistributed in spatially-distinct mitochondrial subpopulations in an inverse manner following diabetic insult. (Abstract shortened by UMI.)

Transcriptome-Wide Characterization of APOBEC1-Catalyzed RNA Editing Events in Macrophages

RNA editing refers to the process by which the sequence of RNA is altered through the insertion, deletion or modification of specific nucleotides. Editing of mRNA transcripts can increase the informational complexity encoded by the genome by producing alternative protein isoforms through specific posttranscriptional RNA editing events. Additionally, RNA editing in non-coding regions of mRNA transcripts has been shown to influence gene expression in a tissue-specific manner. In mammals, mRNA editing serves a diverse set of biological roles in neuronal function, host defense and lipid metabolism. The major mRNA editors acting in mammals include the adenosine deaminases acting on RNA (ADARs) and Apolipoprotein B mRNA Editing Catalytic polypeptide-1 (APOBEC1). The ADARs and APOBEC1 were originally characterized as catalysts for previously characterized biologically important RNA-editing events that resulted in specific coding changes; study of additional editing activity was limited by standard sequencing techniques. APOBEC1 in particular was characterized in the small intestine as mediating a specific editing event in the coding region of Apolipoprotein B (Apob). APOBEC1-dependent RNA editing in Apob mediates the tissue-specific differential expression of Apob isoforms, a process important for intestinal lipid metabolism and transport. The development of next-generation sequencing has allowed for transcriptome-wide discovery of RNA editing activity and has resulted in the identification of more than 10,000 RNA editing events, pointing to more biological functions for RNA editing than had been previously appreciated. To search for additional APOBEC1 editing events, our lab developed a comparative RNA-Seq screen for the transcriptome-wide identification of enzyme-specific RNA editing events. Applying this technique to small intestine enterocytes, the site of known APOBEC1 activity, we identified over 30 novel APOBEC1 editing events in transcript 3’UTRs, which represents the first example of physiological APOBEC1 editing outside of the Apob transcript. These newly identified editing events were located in evolutionarily conserved regions of transcript 3’UTRs, suggesting that this editing activity may have functional relevance. The discovery of additional editing activity for APOBEC1, as well as the fact that it is expressed in a number of immune cell types, suggests that APOBEC1, like other members of the AID/APOBEC family, may contribute to cellular immune processes. The focus of the work presented in this thesis is the identification and characterization of physiological APOBEC1 editing activity in bone marrow derived macrophages (BMDMs). Using a comparative RNA-Seq screen, I identified more than 100 novel APOBEC1 editing events in BMDMs. This APOBEC1 activity occurred in two distinct editing patterns and fell within evolutionarily conserved regions of transcript 3’UTRs. Luciferase reporter assays were utilized to assess the consequences of APOBEC1 3’UTR editing on protein expression and identified a number of combinations of editing events that affect translational outcomes. To determine if APOBEC1 editing could modulate protein expression by altering miRNA targeting, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) of the Argonaute (Ago) proteins was performed on wild-type and APOBEC1-deficient cells. HITS-CLIP yielded transcriptome-wide maps of Ago binding and potential miRNA seed regions. While there was considerable overlap between loci targeted by both Ago and APOBEC1, little evidence was found for APOBEC1 disruption or creation of miRNA seed targets. Overall, this work characterizes abundant APOBEC1 activity in BMDMs that can modulate protein expression levels by a miRNA-independent mechanism. These results point to broader functions for APOBEC1 in transcript regulation and host defense

Transcriptome-Wide Characterization of APOBEC1-Catalyzed RNA Editing Events in Macrophages

RNA editing refers to the process by which the sequence of RNA is altered through the insertion, deletion or modification of specific nucleotides. Editing of mRNA transcripts can increase the informational complexity encoded by the genome by producing alternative protein isoforms through specific posttranscriptional RNA editing events. Additionally, RNA editing in non-coding regions of mRNA transcripts has been shown to influence gene expression in a tissue-specific manner. In mammals, mRNA editing serves a diverse set of biological roles in neuronal function, host defense and lipid metabolism. The major mRNA editors acting in mammals include the adenosine deaminases acting on RNA (ADARs) and Apolipoprotein B mRNA Editing Catalytic polypeptide-1 (APOBEC1). The ADARs and APOBEC1 were originally characterized as catalysts for previously characterized biologically important RNA-editing events that resulted in specific coding changes; study of additional editing activity was limited by standard sequencing techniques. APOBEC1 in particular was characterized in the small intestine as mediating a specific editing event in the coding region of Apolipoprotein B (Apob). APOBEC1-dependent RNA editing in Apob mediates the tissue-specific differential expression of Apob isoforms, a process important for intestinal lipid metabolism and transport. The development of next-generation sequencing has allowed for transcriptome-wide discovery of RNA editing activity and has resulted in the identification of more than 10,000 RNA editing events, pointing to more biological functions for RNA editing than had been previously appreciated. To search for additional APOBEC1 editing events, our lab developed a comparative RNA-Seq screen for the transcriptome-wide identification of enzyme-specific RNA editing events. Applying this technique to small intestine enterocytes, the site of known APOBEC1 activity, we identified over 30 novel APOBEC1 editing events in transcript 3’UTRs, which represents the first example of physiological APOBEC1 editing outside of the Apob transcript. These newly identified editing events were located in evolutionarily conserved regions of transcript 3’UTRs, suggesting that this editing activity may have functional relevance. The discovery of additional editing activity for APOBEC1, as well as the fact that it is expressed in a number of immune cell types, suggests that APOBEC1, like other members of the AID/APOBEC family, may contribute to cellular immune processes. The focus of the work presented in this thesis is the identification and characterization of physiological APOBEC1 editing activity in bone marrow derived macrophages (BMDMs). Using a comparative RNA-Seq screen, I identified more than 100 novel APOBEC1 editing events in BMDMs. This APOBEC1 activity occurred in two distinct editing patterns and fell within evolutionarily conserved regions of transcript 3’UTRs. Luciferase reporter assays were utilized to assess the consequences of APOBEC1 3’UTR editing on protein expression and identified a number of combinations of editing events that affect translational outcomes. To determine if APOBEC1 editing could modulate protein expression by altering miRNA targeting, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) of the Argonaute (Ago) proteins was performed on wild-type and APOBEC1-deficient cells. HITS-CLIP yielded transcriptome-wide maps of Ago binding and potential miRNA seed regions. While there was considerable overlap between loci targeted by both Ago and APOBEC1, little evidence was found for APOBEC1 disruption or creation of miRNA seed targets. Overall, this work characterizes abundant APOBEC1 activity in BMDMs that can modulate protein expression levels by a miRNA-independent mechanism. These results point to broader functions for APOBEC1 in transcript regulation and host defense

Role of miRNAs in Cancer

MicroRNAs are the best representatives of the non-coding part of the genome and their functions are mostly linked to their target genes. During the process of carcinogenesis, both dysregulation of microRNAs and their target genes can explain the development of the disease. However, most of the target genes of microRNAs have not yet been elucidated. In this book, we add new information related to the functions of microRNAs in various tumors and their associated targetome

Post-transcriptional Regulation through Long Noncoding RNAs (lncRNAs)

This book is a collection of eight articles, of which seven are reviews and one is a research paper, that together form a Special Issue that describes the roles that long noncoding RNAs (lncRNA) play in gene regulation at a post-transcriptional level