Biological systems use a variety of mechanisms to maintain their functions in the face of environmental and genetic perturbations. Increasing evidence suggests that, among their roles as ...posttranscriptional repressors of gene expression, microRNAs (miRNAs) help to confer robustness to biological processes by reinforcing transcriptional programs and attenuating aberrant transcripts, and they may in some network contexts help suppress random fluctuations in transcript copy number. These activities have important consequences for normal development and physiology, disease, and evolution. Here, we will discuss examples and principles of miRNAs that contribute to robustness in animal systems.
Significance Cells contain 30â40% dissolved protein and RNA by volume. In vivo protein binding and stability might differ significantly from in vitro measurements. Crowding by the excluded volume ...of these macromolecules has been studied extensively by experiment and theory. When the crowding effects of macromolecules, water, and ions are treated on an equal footing, the effect is opposite to that commonly believed. Large molecules are less effective at crowding than water and ions. There is also a surprisingly weak dependence on crowder size. Molecules of medium size have the same effect as much larger macromolecules like proteins and RNA. These results require a reassessment of observed high-concentration effects and of strategies to mimic in vivo conditions with in vitro experiments.
The aqueous milieu inside cells contains as much as 30â40% dissolved protein and RNA by volume. This large concentration of macromolecules is expected to cause significant deviations from solution ideality. In vivo biochemical reaction rates and equilibria might differ significantly from those measured in the majority of in vitro experiments that are performed at much lower macromolecule concentrations. Consequently crowding, a nonspecific phenomenon believed to arise from the large excluded volume of these macromolecules, has been studied extensively by experimental and theoretical methods. However, the relevant theory has not been applied consistently. When the steric effects of macromolecular crowders and small molecules like water and ions are treated on an equal footing, the effect of the macromolecules is opposite to that commonly believed. Large molecules are less effective at crowding than water and ions. There is also a surprisingly weak dependence on crowder size. Molecules of medium size, â¼5 AÌ radius, have the same effect as much larger macromolecules like proteins and RNA. These results require a reassessment of observed high-concentration effects and of strategies to mimic in vivo conditions with in vitro experiments.
The mammalian genome is extensively transcribed, a large fraction of which is divergent transcription from promoters and enhancers that is tightly coupled with active gene transcription. Here, we ...propose that divergent transcription may shape the evolution of the genome by new gene origination.
Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA ...networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of in vivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict in vivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ∼3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition in vivo.
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•Quantitation of miRNA and iCLIP-supported target pool copies per cell for all miRNAs•Low miRNA:target ratio miRNAs predominantly bind high-affinity sites•High-affinity targets of low abundance miRNAs are susceptible to high-affinity ceRNA•High-abundance miRNAs are not susceptible to ceRNA titration in physiological ranges
Bosson et al. estimate the total copies per cell of all miRNA and corresponding targets to address the debated competing endogenous RNA (ceRNA) hypothesis. They show that only miRNAs with low miRNA:target ratios are susceptible to regulation by large induction of high-affinity ceRNAs.
Phase-separated multi-molecular assemblies provide a general regulatory mechanism to compartmentalize biochemical reactions within cells. We propose that a phase separation model explains established ...and recently described features of transcriptional control. These features include the formation of super-enhancers, the sensitivity of super-enhancers to perturbation, the transcriptional bursting patterns of enhancers, and the ability of an enhancer to produce simultaneous activation at multiple genes. This model provides a conceptual framework to further explore principles of gene control in mammals.
A phase separation model for transcription explains key features of transcription and sets enhancers, and especially super-enhancers, into the broad family of membraneless organelles.
Most genetic information is expressed as, and transacted by, proteins. Yet, less than 2% of the human genome actually codes for proteins, prompting a search for functions for the other 98% of the ...genome, once considered to be mostly "junk DNA." Transcription is pervasive, however, and high-throughput sequencing has identified tens of thousands of distinct RNAs generated from the non-protein-coding portion of the genome (1). These so-called noncoding RNAs vary in length, but like protein-coding RNAs, appear to be linear molecules with 5' and 3' termini, reflecting the defined start and end points of RNA polymerase on the DNA template. But do all RNAs have to be linear?
The Centrality of RNA Sharp, Phillip A.
Cell,
02/2009, Volume:
136, Issue:
4
Journal Article
Peer reviewed
Open access
New roles for RNAs in biology continue to emerge, and a glance at the history of RNAs may prepare molecular biologists for future discoveries about these powerful molecules. A striking new role for ...RNAs is their widespread involvement in the regulation of numerous genes, suggesting that there is much yet to discover about these amazing cellular components.
Specific protein-RNA interactions guide posttranscriptional gene regulation. Here, we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity ...of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1, and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity.
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•RBNS is a method for comprehensive, quantitative mapping of RNA binding specificity•RBNS identifies multiple motifs recognized by RBFOX2, CELF1, and MBNL1 proteins•RNA structure inhibits binding, except MBNL1 tolerates G/C pairing in UGC motifs•RBNS distinguishes subsets of functional and nonfunctional CLIP-seq motifs
Lambert et al. develop a sequencing-based method to determine the sequence and structural specificity of RNA binding proteins (RBPs). They identify new motifs for three RBPs, detect effects of RNA structure on binding, and distinguish the subset of motifs identified by UV crosslinking that have regulatory activity.
Liver fibrosis is characterised by excessive deposition of extracellular matrix that interrupts normal liver functionality. It is a pathological stage in several untreated chronic liver diseases such ...as the iron overload syndrome hereditary haemochromatosis, viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and diabetes. Interestingly, regardless of the aetiology, iron-loading is frequently observed in chronic liver diseases. Excess iron can feed the Fenton reaction to generate unquenchable amounts of free radicals that cause grave cellular and tissue damage and thereby contribute to fibrosis. Moreover, excess iron can induce fibrosis-promoting signals in the parenchymal and non-parenchymal cells, which accelerate disease progression and exacerbate liver pathology. Fibrosis regression is achievable following treatment, but if untreated or unsuccessful, it can progress to the irreversible cirrhotic stage leading to organ failure and hepatocellular carcinoma, where resection or transplantation remain the only curative options. Therefore, understanding the role of iron in liver fibrosis is extremely essential as it can help in formulating iron-related diagnostic, prognostic and treatment strategies. These can be implemented in isolation or in combination with the current approaches to prepone detection, and halt or decelerate fibrosis progression before it reaches the irreparable stage. Thus, this review narrates the role of iron in liver fibrosis. It examines the underlying mechanisms by which excess iron can facilitate fibrotic responses. It describes the role of iron in various clinical pathologies and lastly, highlights the significance and potential of iron-related proteins in the diagnosis and therapeutics of liver fibrosis.
MicroRNAs (miRNAs) are key regulators of gene expression. They are conserved across species, expressed across cell types, and active against a large proportion of the transcriptome. The ...sequence-complementary mechanism of miRNA activity exploits combinatorial diversity, a property conducive to network-wide regulation of gene expression, and functional evidence supporting this hypothesized systems-level role has steadily begun to accumulate. The emerging models are exciting and will yield deep insight into the regulatory architecture of biology. However, because of the technical challenges facing the network-based study of miRNAs, many gaps remain. Here, we review mammalian miRNAs by describing recent advances in understanding their molecular activity and network-wide function.
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► Network-level perspective of miRNA activity. ► A summary of the structure of eukaryotic Argonaute. ► A comparison of miRNA function in vitro versus in vivo. ► A description of four miRNA families: miR-290–295, let-7, miR-17–92, and miR-34.
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