Genes come and go: The evolutionarily plastic path of budding yeast RNase III enzymes
Our recent finding that the Candida albicans RNase III enzyme CaDcr1 is an unusual, multifunctional RNase III coupled with data on the RNase III enzymes from other fungal species prompted us to seek a model that explained the evolution of RNase III’s in modern budding yeast species. CaDcr1 has both dicer function (generates small RNA molecules from dsRNA precursors) and Rnt1 function, (catalyzes the maturation of 35S rRNA and U4 snRNA). Some budding yeast species have two distinct genes that encode these functions, a Dicer and RNT1, whereas others have only an RNT1 and no Dicer. As none of the budding yeast species has the canonical Dicer found in many other fungal lineages and most eukaryotes, the extant species must have evolved from an ancestor that lost the canonical Dicer, and evolved a novel Dicer from the essential RNT1 gene. No single, simple model could explain the evolution of RNase III enzymes from this ancestor because existing sequence data are consistent with two equally plausible models. The models share an architecture for RNase III evolution that involves gene duplication, loss, subfunctionalization, and neofunctionalization. This commentary explains our reasoning, and offers the prospect that further genomic data could further resolve the dilemma surrounding the budding yeast RNase III’s evolution.
Emerging roles for Sam68 in adipogenesis and neuronal development
Sam68, the Src-associated substrate during mitosis of 68 kDa, belongs to the large class of heteronuclear ribonucleoprotein particle K (hnRNP K) homology (KH) domain family of RNA-binding proteins. Sam68 contains a single KH domain harboring conserved N- and C-terminal sequences required for RNA binding and homodimerization. The KH domain is one of the most prevalent RNA binding domains that directly contacts single-stranded RNA. Sam68 has been implicated in numerous aspects of RNA metabolism including alternative splicing and polysomal recruitment of mRNAs. Studies in mice have revealed physiological roles linking Sam68 to osteoporosis, obesity, cancer, infertility and ataxia. Recent publications have greatly enhanced our understanding of Sam68 mechanism of action in addition to its cellular role. Herein, we will discuss the latest advances in the literature pertaining to obesity and neuronal development.
Cascade-mediated binding and bending of negatively supercoiled DNA
Prokaryotes possess various defense mechanisms against invading DNA. Adaptive defense by CRISPR/Cas relies on incorporation of invader DNA sequences in the host genome. In Escherichia coli, processed transcripts of these incorporated sequences (crRNAs) guide Cascade-mediated invader DNA recognition.1–4 Cascade is a multisubunit ribonucleoprotein complex, consisting of one crRNA and five proteins: Cse1, Cse2, Cas7, Cas5 and Cas6e.1, 2 Cascade-mediated DNA recognition requires a conserved sequence adjacent to the target (protospacer adjacent motif, PAM) and a negatively supercoiled DNA topology.3, 4 While Cse1 carries out PAM recognition,5 the Cascade structure suggests that Cse2 may interact with target DNA in the PAM-distal end of the protospacer.6 Using Electrophoretic Mobility Shift Assays, we here describe the function of the Cse1 and Cse2 subunits in the context of protospacer recognition on negatively supercoiled DNA. While Cse1 is required for nonspecific DNA binding, Cse2 appears to be important for specific binding, presumably by mediating stabilizing interactions with the displaced strand, the R-loop, or both. Furthermore, we performed Scanning Force Microscopy using linearized DNA molecules, which facilitates accurate and reliable measurements of Cascade-mediated bending. This analysis reveals that Cascade binding induces flexibility in the DNA target, most likely due to single stranded DNA regions flanking the R-loop.
Telomerase caught in the act: United we stand, divided we fall
The stable linearity of eukaryotic chromosomes depends on special characteristics of their ends, the telomeres. Accurate telomere function in turn requires a sustained presence of repeated DNA elements, which are maintained by the enzyme telomerase. The telomerase holoenzyme is composed of both protein and RNA, and its functions rely on proper expression, maturation, trafficking and assembly of these components. Conflicting models for the recruitment of telomerase at telomeres have been proposed; one suggests a local activation of telomerase at short telomeres, while the other proposes that telomerase is recruited only at short telomeres. To discriminate between these models and investigate the cell cycle-dependent regulation of telomerase in living cells, a GFP reporter system to visualize the yeast telomerase RNA has been recently developed. This assay shed new light on the mechanism of recruitment of telomerase to telomeres, and it uncovered a hitherto unrecognized mechanism for restricting telomerase access to telomeres.
Tyrosine-1 and threonine-4 phosphorylation marks complete the RNA polymerase II CTD phospho-code
Eukaryotic RNA polymerase II (RNAP II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of its largest subunit (Rpb1). Dynamic phosphorylation of Ser2, Ser5 and Ser7 residues orchestrates the binding of transcription and RNA processing factors to the transcription machinery. Recent studies show that the two remaining potential phosphorylation sites, tyrosine-1 and threonine-4, are phosphorylated as well and contribute to the previously proposed “CTD code“. With the impairment of binding of CTD interacting factors, these novel phosphorylation marks add an accessory layer of regulation to the RNAP II transcription cycle.
XenomiRs and miRNA homeostasis in health and disease: Evidence that diet and dietary miRNAs directly and indirectly influence circulating miRNA profiles
Contributions of dietary miRNAs to circulating small RNA profiles would have profound implications for interpretation of miRNA biomarker studies: presumptive disease-specific markers might instead indicate responses to disease-associated quantitative or qualitative dietary alteration. This examination weighs the evidence for a 2-fold hypothesis: first, that ingested biological matter contributes directly to the miRNA complement of body compartments; and second, that these diet-derived exogenous miRNAs (or “xenomiRs”) affect total miRNA profiles as part of a circulating miRNA homeostasis that is altered in many diseases. Homeostasis of high-density lipoprotein (HDL), a known miRNA carrier—provides a model as a proposed component of broader miRNA homeostasis. Further research into the dietary xenomiR hypothesis is needed to ensure rigor in the search for truly disease-specific miRNA biomarkers.
The RNA component of the RNase P complex is found throughout most branches of the tree of life and is principally responsible for removing the 5′ leader sequence from pre-tRNA transcripts during tRNA maturation. RNase P RNA has a number of universal core features, however variations in sequence and structure found in homologs across the tree of life require multiple Rfam covariance search models to detect accurately. We describe a new Rfam search model to enable efficient detection of the diminutive archaeal Type T RNase P RNAs, which are missed by existing Rfam models. Using the new model, we establish effective score detection thresholds, and detect four new RNase P RNA genes in recently completed genomes from the crenarchaeal family Thermoproteaceae.
The mitochondrial genome of metazoan animal typically encodes 22 tRNAs. Nematode mt-tRNAs normally lack the T-stem and instead feature a replacement loop. In the class Enoplea, putative mt-tRNAs that are even further reduced have been predicted to lack both the T- and the D-arm. Here we investigate these tRNA candidates in detail. Three lines of computational evidence support that they are indeed minimal functional mt-tRNAs: (1) the high level of conservation of both sequence and secondary structure, (2) the perfect preservation of the anticodons, and (3) the persistence of these sequence elements throughout several genome rearrangements that place them between different flanking genes.
A novel fluorescent reporter system for monitoring and identifying RNase III activity and its target RNAs
Bacteriophage vectors for achieving single-copy gene expression linked to a colorigenic reporter assay have been used successfully for genetic screening applications. However, the limited number of cloning sites in these vectors, combined with the requirement for lac- strains and the time- and/or media-dependence of the chemical-based colorimetric reaction, have limited the range of applications for these vectors. An alternative approach using a fluorescent reporter gene such as green fluorescent protein (GFP) or GFP derivatives could overcome some of these technical issues and facilitate real-time monitoring of promoter and/or protein activity. Here, we report the development of a novel translational bacteriophage fusion vector encoding enhanced GFP (eGFP) that can be incorporated into the chromosome as a single-copy gene. We identified a Bacillus promoter (BP) that is stably expressed in Escherichia coli and drives ~6-fold more expression of eGFP than the T7 promoter in the absence of inducer. Incorporating this BP and RNase III target signals into a single system enabled clear detection of the absence or downregulation of RNase III activity in vivo, thereby establishing a system for screening and identifying novel RNase III targets in a matter of days. An RNase III target signal identified in this manner was confirmed by post-transcriptional analysis. We anticipate that this novel translational fusion vector will be used extensively to study activity of both interesting RNases and related complex or to identify or validate targets of RNases that are otherwise difficult to study due to their sensitivity to environmental stresses and/or autoregulatory processes.
Human mirtrons can express functional microRNAs simultaneously from both arms in a flanking exon-independent manner
Mirtrons are short intronic microRNA (miRNA) precursors representing an alternative, Drosha/DGCR8-independent miRNA biogenesis pathway. In this study we characterized three predicted human mirtrons. Their expression was proven to be context-independent, since functional mirtrons could be derived either from their endogenous or from a heterologous coding environment. Systematic testing revealed that both 5′- and 3′-arms of mir-877 are capable of producing functional miRNA simultaneously in the various cell types examined. On the other hand, experimental validations revealed that the predicted mir-1233 is not a bona fide mirtron. For functional mirtrons, we were able to detect mature mirtron-derived miRNAs for the first time by qRT-PCR or northern blot analysis, when silencing activity was proven by functional assays. Our results emphasize the need for functional testing of both arms of miRNAs and the importance of experimentally validating human mirtrons since, in spite of being localized in a short intron, predicted species could mature via other miRNA processing pathways.
Ribonuclease P-mediated inhibition of human cytomegalovirus gene expression and replication induced by engineered external guide sequences
External guide sequences (EGSs) are RNA molecules that can bind to a target mRNA and direct ribonuclease P (RNase P), a tRNA processing enzyme, for specific cleavage of the target mRNA. Using an in vitro selection procedure, we have previously generated EGS variants that efficiently direct human RNase P to cleave a target mRNA in vitro. In this study, we constructed EGSs from a variant to target the overlapping region of the mRNAs coding for human cytomegalovirus (HCMV) capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The EGS variant was about 40-fold more active in directing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of about 98% and 75% in CSP/assemblin gene expression and a reduction of 7000- and 250-fold in viral growth were observed in HCMV-infected cells that expressed the variant and the tRNA-derived EGS, respectively. Our study shows that the EGS variant is more effective in blocking HCMV gene expression and growth than the tRNA-derived EGS. Moreover, these results demonstrate the utility of highly active EGS RNA variants in gene targeting applications including anti-HCMV therapy.
A new microRNA target prediction tool identifies a novel interaction of a putative miRNA with CCND2
Computational methods for miRNA target prediction vary in the algorithm used; and while one can state opinions about the strengths or weaknesses of each particular algorithm, the fact of the matter is that they fall substantially short of capturing the full detail of physical, temporal and spatial requirements of miRNA::target-mRNA interactions. Here, we introduce a novel miRNA target prediction tool called Targetprofiler that utilizes a probabilistic learning algorithm in the form of a hidden Markov model trained on experimentally verified miRNA targets. Using a large scale protein downregulation data set we validate our method and compare its performance to existing tools. We find that Targetprofiler exhibits greater correlation between computational predictions and protein downregulation and predicts experimentally verified miRNA targets more accurately than three other tools. Concurrently, we use primer extension to identify the mature sequence of a novel miRNA gene recently identified within a cancer associated genomic region and use Targetprofiler to predict its potential targets. Experimental verification of the ability of this small RNA molecule to regulate the expression of CCND2, a gene with documented oncogenic activity, confirms its functional role as a miRNA. These findings highlight the competitive advantage of our tool and its efficacy in extracting biologically significant results.