The Significance of the DUF283 Domain for the Activity of Human Ribonuclease Dicer

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.     

Archaeal-bacterial chimeric RNase P RNAs: towards understanding RNA's architecture, function and evolution

The higher protein content of archaeal RNase P (1 RNA+4 proteins) compared to the bacterial homologue (1 RNA+1 protein) correlates with a large loss of RNA-alone activity (i.e., in the absence of protein cofactors). Here we show, for the first time, that a catalytic (C) domain of an archaeal RNase P RNA (P RNA) can functionally replace the Escherichia coli C domain in a chimeric P RNA, to provide the essential RNase P function in E. coli cells. This adaptation was achieved by 1) three minor alterations in the archaeal C domain, 2) restoration of the L9-P1 interdomain contact that is found in bacterial and archaeal type A RNAs, and 3) installation of another interdomain contact (L18-P8) that is present in bacterial but absent in archaeal P RNAs. We conclude 1) that the C domains of bacterial and archaeal P RNAs of type A have been largely conserved since the evolutionary separation of bacteria and archaea, and 2) that the L18-P8 RNA-RNA contact has been replaced with protein-protein contacts in archaeal RNase P. Function of the chimeric P RNA in E. coli required overexpression of the E. coli RNase P protein to increase the RNA's reduced cellular levels; this was attributed to enhanced degradation of the chimeric P RNA.     

The Evolutionary Significance of RNAi in the Fungal Kingdom

RNA interference (RNAi) was discovered at the end of last millennium, changing the way scientists understood regulation of gene expression. Within the following two decades, a variety of different RNAi mechanisms were found in eukaryotes, reflecting the evolutive diversity that RNAi entails. The essential silencing mechanism consists of an RNase III enzyme called Dicer that cleaves double-stranded RNA (dsRNA) generating small interfering RNAs (siRNAs), a hallmark of RNAi. These siRNAs are loaded into the RNA-induced silencing complex (RISC) triggering the cleavage of complementary messenger RNAs by the Argonaute protein, the main component of the complex. Consequently, the expression of target genes is silenced. This mechanism has been thoroughly studied in fungi due to their proximity to the animal phylum and the conservation of the RNAi mechanism from lower to higher eukaryotes. However, the role and even the presence of RNAi differ across the fungal kingdom, as it has evolved adapting to the particularities and needs of each species. Fungi have exploited RNAi to regulate a variety of cell activities as different as defense against exogenous and potentially harmful DNA, genome integrity, development, drug tolerance, or virulence. This pathway has offered versatility to fungi through evolution, favoring the enormous diversity this kingdom comprises.     

ADAR Enzyme and miRNA Story: A Nucleotide that Can Make the Difference

Adenosine deaminase acting on RNA (ADAR) enzymes convert adenosine (A) to inosine (I) in double-stranded (ds) RNAs. Since Inosine is read as Guanosine, the biological consequence of ADAR enzyme activity is an A/G conversion within RNA molecules. A-to-I editing events can occur on both coding and non-coding RNAs, including microRNAs (miRNAs), which are small regulatory RNAs of ~20–23 nucleotides that regulate several cell processes by annealing to target mRNAs and inhibiting their translation. Both miRNA precursors and mature miRNAs undergo A-to-I RNA editing, affecting the miRNA maturation process and activity. ADARs can also edit 3′ UTR of mRNAs, further increasing the interplay between mRNA targets and miRNAs. In this review, we provide a general overview of the ADAR enzymes and their mechanisms of action as well as miRNA processing and function. We then review the more recent findings about the impact of ADAR-mediated activity on the miRNA pathway in terms of biogenesis, target recognition, and gene expression regulation.     

Mechanisms of Lin28-Mediated miRNA and mRNA Regulation—A Structural and Functional Perspective

Lin28 is an essential RNA-binding protein that is ubiquitously expressed in embryonic stem cells. Its physiological function has been linked to the regulation of differentiation, development, and oncogenesis as well as glucose metabolism. Lin28 mediates these pleiotropic functions by inhibiting let-7 miRNA biogenesis and by modulating the translation of target mRNAs. Both activities strongly depend on Lin28’s RNA-binding domains (RBDs), an N-terminal cold-shock domain (CSD) and a C-terminal Zn-knuckle domain (ZKD). Recent biochemical and structural studies revealed the mechanisms of how Lin28 controls let-7 biogenesis. Lin28 binds to the terminal loop of pri- and pre-let-7 miRNA and represses their processing by Drosha and Dicer. Several biochemical and structural studies showed that the specificity of this interaction is mainly mediated by the ZKD with a conserved GGAGA or GGAGA-like motif. Further RNA crosslinking and immunoprecipitation coupled to high-throughput sequencing (CLIP-seq) studies confirmed this binding motif and uncovered a large number of new mRNA binding sites. Here we review exciting recent progress in our understanding of how Lin28 binds structurally diverse RNAs and fulfills its pleiotropic functions.     

Molecular mechanisms of RNA-triggered gene silencing machineries

Gene silencing by RNA triggers is an ancient, evolutionarily conserved, and widespread phenomenon. This process, known as RNA interference (RNAi), occurs when double-stranded RNA helices induce cleavage of their complementary mRNAs. Because these RNA molecules can be introduced exogenously as small interfering RNAs (siRNAs), RNAi has become an everyday experimental tool in laboratory research. In addition, the number of RNA-based therapeutics that are currently in clinical trials for a variety of human diseases demonstrate the therapeutic potential of RNAi. In this Account, we focus on our current understanding of the structure and function of various classes of RNAi triggers and how this knowledge has contributed to our understanding of the biogenesis and catalytic functions of siRNA and microRNA in mammalian cells. Mechanistic studies to understand the structure and function of small RNAs that induce RNAi have illuminated broad functions of the ancient RNAi machinery in animals and plants. In addition, such studies have provided insight to identify endogenous physiological gene silencing RNA triggers that engage functional machineries similar to siRNAs. Several endogenous small RNA species have been identified: small noncoding RNAs (microRNAs), piwi-interacting RNAs (piRNAs), and endogenous siRNAs (endo-siRNAs). microRNAs are the most widespread class of small RNAs in mammalian cells. Despite their importance in biology and medicine, the molecular and cellular mechanisms of microRNA biogenesis and function are not fully understood. We provide an overview of the current understanding of how these molecules are synthesized within cells and how they act on gene targets. Interesting questions remain both for understanding the effects of modifications and editing on microRNAs and the interactions between microRNAs and other cellular RNAs such as long noncoding RNAs.     

Non-Coding RNA-Driven Regulation of rRNA Biogenesis

Ribosomal RNA (rRNA) biogenesis takes place in the nucleolus, the most prominent condensate of the eukaryotic nucleus. The proper assembly and integrity of the nucleolus reflects the accurate synthesis and processing of rRNAs which in turn, as major components of ribosomes, ensure the uninterrupted flow of the genetic information during translation. Therefore, the abundant production of rRNAs in a precisely functional nucleolus is of outmost importance for the cell viability and requires the concerted action of essential enzymes, associated factors and epigenetic marks. The coordination and regulation of such an elaborate process depends on not only protein factors, but also on numerous regulatory non-coding RNAs (ncRNAs). Herein, we focus on RNA-mediated mechanisms that control the synthesis, processing and modification of rRNAs in mammals. We highlight the significance of regulatory ncRNAs in rRNA biogenesis and the maintenance of the nucleolar morphology, as well as their role in human diseases and as novel druggable molecular targets.     

RNase L Cleavage Products Promote Switch from Autophagy to Apoptosis by Caspase-Mediated Cleavage of Beclin-1

Autophagy and apoptosis share regulatory molecules enabling crosstalk in pathways that affect cellular homeostasis including response to viral infections and survival of tumor cells. Ribonuclease L (RNase L) is an antiviral endonuclease that is activated in virus-infected cells and cleaves viral and cellular single-stranded RNAs to produce small double-stranded RNAs with roles in amplifying host responses. Activation of RNase L induces autophagy and apoptosis in many cell types. However, the mechanism by which RNase L mediates crosstalk between these two pathways remains unclear. Here we show that small dsRNAs produced by RNase L promote a switch from autophagy to apoptosis by caspase-mediated cleavage of Beclin-1, terminating autophagy. The caspase 3-cleaved C-terminal fragment of Beclin-1 enhances apoptosis by translocating to the mitochondria along with proapoptotic protein, Bax, and inducing release of cytochrome C to the cytosol. Cleavage of Beclin-1 determines switch to apoptosis since expression of caspase-resistant Beclin-1 inhibits apoptosis and sustains autophagy. Moreover, inhibiting RNase L-induced autophagy promotes cell death and inhibiting apoptosis prolongs autophagy in a cross-inhibitory mechanism. Our results demonstrate a novel role of RNase L generated small RNAs in cross-talk between autophagy and apoptosis that impacts the fate of cells during viral infections and cancer.     

Evaluation of bacterial RNase P RNA as a drug target

RNA has gained increasing importance as a therapeutic target. However, so far mRNAs rather than stable cellular RNAs have been considered in such studies. In bacteria, the tRNA-processing enzyme RNase P has a catalytic RNA subunit. Fundamental differences in structure and function between bacterial and eukaryotic RNase P, and its indispensability for cell viability make the bacterial enzyme an attractive drug target candidate. Herein we describe two approaches utilized to evaluate whether the catalytic RNA subunit of bacterial RNase P is amenable to inactivation by antisense-based strategies. In the first approach, we rationally designed RNA hairpin oligonucleotides targeted at the tRNA 3'-CCA binding site (P15 loop region) of bacterial RNase P RNA by attempting to include principles derived from the natural CopA-CopT antisense system. Substantial inactivation of RNase P RNA was observed for Type A RNase P RNA (such as that in Escherichia coli) but not for Type B (as in Mycoplasma hyopneumoniae). Moreover, only an RNA oligonucleotide (Eco 3') complementary to the CCA binding site and its 3' flanking sequences was shown to be an efficient inhibitor. Mutation of Eco 3' and analysis of other natural RNase P RNAs with sequence deviations in the P15 loop region showed that inhibition is due to interaction of Eco 3' with this region and occurs in a highly sequence-specific manner. A DNA version of Eco 3' was a less potent inhibitor. The potential of Eco 3' to form an initial kissing complex with the P15 loop did not prove advantageous. In a second approach, we tested a set of oligonucleotides against E. coli RNase P RNA which were designed by algorithms developed for the selection of suitable mRNA targets. This approach identified the P10/11-J11/12 region of bacterial RNase P RNA as another accessible region. In conclusion, both the P15 loop and P10/11-J11/12 regions of Type A RNase P RNAs seem to be promising antisense target sites since they are easily accessible and sufficiently interspersed with nonhelical sequence elements, and oligonucleotide binding directly interferes with substrate docking to these two regions.     

Ribosome Biogenesis and Cancer: Overview on Ribosomal Proteins

Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.     

SRPassing Co-translational Targeting: The Role of the Signal Recognition Particle in Protein Targeting and mRNA Protection

Signal recognition particle (SRP) is an RNA and protein complex that exists in all domains of life. It consists of one protein and one noncoding RNA in some bacteria. It is more complex in eukaryotes and consists of six proteins and one noncoding RNA in mammals. In the eukaryotic cytoplasm, SRP co-translationally targets proteins to the endoplasmic reticulum and prevents misfolding and aggregation of the secretory proteins in the cytoplasm. It was demonstrated recently that SRP also possesses an earlier unknown function, the protection of mRNAs of secretory proteins from degradation. In this review, we analyze the progress in studies of SRPs from different organisms, SRP biogenesis, its structure, and function in protein targeting and mRNA protection.     

α-Helices in the Type III Secretion Effectors: A Prevalent Feature with Versatile Roles

Type III Secretion Systems (T3SSs) are multicomponent nanomachines located at the cell envelope of Gram-negative bacteria. Their main function is to transport bacterial proteins either extracellularly or directly into the eukaryotic host cell cytoplasm. Type III Secretion effectors (T3SEs), latest to be secreted T3S substrates, are destined to act at the eukaryotic host cell cytoplasm and occasionally at the nucleus, hijacking cellular processes through mimicking eukaryotic proteins. A broad range of functions is attributed to T3SEs, ranging from the manipulation of the host cell’s metabolism for the benefit of the bacterium to bypassing the host’s defense mechanisms. To perform this broad range of manipulations, T3SEs have evolved numerous novel folds that are compatible with some basic requirements: they should be able to easily unfold, pass through the narrow T3SS channel, and refold to an active form when on the other side. In this review, the various folds of T3SEs are presented with the emphasis placed on the functional and structural importance of α-helices and helical domains.     

The GI-UEV Domain,a Catalytically Inactive Ubiquitin-Conjugating Enzyme Variant With a Role in Translational Regulation

Ubiquitin-conjugating enzymes (UBCs) form the second step in the enzyme cascade required for protein ubiquitylation. Most eukaryotic genomes contain a multitude of different catalytically active UBCs. In addition, several proteins contain homology domains related to UBCs that have lost their catalytic activity and are referred to as “ubiquitin-conjugating enzyme variants” or UEV-domains. A common property of those domains is a role in ubiquitin binding and recognition. We report here on a novel class of more distantly related UEV domains, which forms a superset of the previously described GI-homology region found in Gcn2 and IMPACT proteins. In the Gcn2 and IMPACT protein families, the GI-UEV domain binds to proteins of the Gcn1 family and thus regulates translation levels via eIF2α phosphorylation. Bioinformatical analysis shows that GI-UEV domains occur in a large number of proteins, many of them without an established role in translational regulation. Residue conservation and domain context predict that GI-UEV domains might also have a role in the ubiquitin/proteasome system, suggesting a possible cross-talk between ubiquitylation and translational regulation.