SUMMARY MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3′UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.

SsrA is a versatile RNA molecule found in all bacteria that functions as both a tRNA and an mRNA. SsrA rescues ribosomes stalled on damaged mRNAs and directs the tagging and degradation of their aberrant protein products. Small protein B (SmpB) is required for all known activities of SsrA. The two known functions of SmpB are binding SsrA RNA and promoting stable association of the SmpB⅐SsrA complex with 70S ribosomes. Using mutational analysis and biochemical experiments, we have discovered a previously uncharacterized SmpB function. This function is required for a step in the tagging process downstream of SsrA binding and ribosome association but before transpeptidation of the SsrA-linked alanine and establishment of the SsrA reading frame. Our results clearly demonstrate that residues in the C-terminal tail of SmpB confer a hitherto unrevealed function that is essential for trans-translation. Based on these results, we propose that upon binding stalled ribosomes, the unstructured C-terminal tail of SmpB acquires contacts that are critical for productive accommodation of SsrA into the ribosomal A site.SsrA ͉ translation

The accurate flow of genetic information from DNA to RNA to protein is essential for all living organisms. An astonishing array of quality-assurance mechanisms have evolved to ensure that high degree of fidelity is maintained at every stage of this process. One of the most fascinating quality-control mechanisms involves tmRNA, also known as SsrA or 10Sa RNA. tmRNA is a versatile and highly conserved bacterial molecule endowed with the combined structural and functional properties of both a tRNA and a mRNA. The tmRNA system orchestrates three key biological functions: (1) recognition and rescue of ribosomes stalled on aberrant mRNAs, (2) disposal of the causative defective mRNAs, and (3) addition of a degradation tag to ribosome-associated protein fragments for directed proteolysis. Although not essential in Escherichia coli, tmRNA activity is essential for bacterial survival under adverse conditions and for virulence in some, and perhaps all, pathogenic bacteria. Recent evidence suggests that in addition to its quality-control function the tmRNA system might also play a key regulatory role in certain physiological pathways. This review will focus on recent advances in our understanding of the structural properties, mechanistic details, and physiological significance of this unique RNA and its principal protein partners.

Photoreceptor cell death is the proximal cause of blindness in many retinal degenerative disorders; hence, understanding the gene regulatory networks that promote photoreceptor survival is at the forefront of efforts to combat blindness. Down-regulation of the microRNA (miRNA)-processing enzyme DICER1 in the retinal pigmented epithelium has been implicated in geographic atrophy, an advanced form of age-related macular degeneration (AMD). However, little is known about the function of DICER1 in mature rod photoreceptor cells, another retinal cell type that is severely affected in AMD. Using a conditional-knockout (cKO) mouse model, we report that loss of DICER1 in mature postmitotic rods leads to robust retinal degeneration accompanied by loss of visual function. At 14 wk of age, cKO mice exhibit a 90% reduction in photoreceptor nuclei and a 97% reduction in visual chromophore compared with those in control littermates. Before degeneration, cKO mice do not exhibit significant defects in either phototransduction or the visual cycle, suggesting that miRNAs play a primary role in rod photoreceptor survival. Using comparative small RNA sequencing analysis, we identified rod photoreceptor miRNAs of the miR-22, miR-26, miR-30, miR-92, miR-124, and let-7 families as potential factors involved in regulating the survival of rods.

The discovery of microRNA (miRNA)-mediated gene silencing has added a new level of complexity to our understanding of post-transcriptional control of gene expression. Considering the ubiquity of miRNA-mediated repression throughout basic cellular processes, understanding its mechanism of action is paramount to obtain a clear picture of the regulation of gene expression in biological systems. Although many miRNAs and their targets have been identified, a detailed understanding of miRNA action remains elusive. miRNAs regulate gene expression at the post-transcriptional level, through both translational inhibition and mRNA destabilization. Recent reports suggest that many miRNA effects are mediated through proteins of the GW182 family. This chapter focuses on the multiple and potentially overlapping mechanisms that miRNAs utilize to regulate gene expression in eukaryotes.

microRNAs (miRNAs) are small, stable RNA molecules that post-transcriptionally regulate gene expression in plants and animals by base pairing to partially complementary sequences on target mRNAs to inhibit protein synthesis. More than 250 miRNAs are reportedly expressed in the retina, and miRNA gene regulation has been shown to affect retinal development, function, and disease. Here we highlight recent advances in understanding the functional roles of vertebrate retinal miRNAs. Details are emerging about the physiological impact of specific miRNAs in the developing and mature retina, and we discuss a group of emerging technologies for studying miRNAs, which can be employed to yield a deeper understanding of retinal miRNA gene regulation.

MicroRNAs (miRNAs) regulate multiple genes, often within the same pathway, fine-tuning expression of key factors and stabilizing gene networks against aberrant fluctuations. The demanding physiologic functions of photoreceptor cells and the retinal pigmented epithelium necessitate precise gene regulation to maintain their homeostasis and function, thus rendering these postmitotic cells vulnerable to premature death in retinal degenerative disorders. Recent studies of the physiologic impact of miRNAs in these cells clearly demonstrate that miRNAs are an essential component of that gene regulation. These important advances provide the foundation for future exploration of miRNA-regulated gene networks in the eye to facilitate the development of miRNA-targeted therapeutics to combat blinding diseases.-Sundermeier, T. R., Palczewski, K. The impact of microRNA gene regulation on the survival and function of mature cell types in the eye. FASEB J. 30, 23-33 (2016). www.fasebj.org

BackgroundThough accumulating evidence suggests that microglia, resident macrophages in the retina, and bone marrow-derived macrophages can cause retinal inflammation which accelerates photoreceptor cell death, the details of how these cells are activated during retinal degeneration (RD) remain uncertain. Therefore, it is important to clarify which cells play a dominant role in fueling retinal inflammation. However, distinguishing between microglia and macrophages is difficult using conventional techniques such as cell markers (e.g., Iba-1). Recently, two mouse models for visualizing chemokine receptors were established, Cx3cr1GFP/GFP and Ccr2RFP/RFP mice. As Cx3cr1 is expressed in microglia and Ccr2 is reportedly expressed in activated macrophages, these mice have the potential to distinguish microglia and macrophages, yielding novel information about the activation of these inflammatory cells and their individual roles in retinal inflammation.MethodsIn this study, c-mer proto-oncogene tyrosine kinase (Mertk)−/− mice, which show photoreceptor cell death due to defective retinal pigment epithelium phagocytosis, were employed as an animal model of RD. Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice were established by breeding Mertk−/−, Cx3cr1GFP/GFP, and Ccr2RFP/RFP mice. The retinal morphology and pattern of inflammatory cell activation and invasion of Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice were evaluated using retina and retinal pigment epithelium (RPE) flat mounts, retinal sections, and flow cytometry.ResultsFour-week-old Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice showed Cx3cr1-GFP-positive microglia in the inner retina. Cx3cr1-GFP and Ccr2-RFP dual positive activated microglia were observed in the outer retina and subretinal space of 6- and 8-week-old animals. Ccr2-RFP single positive bone marrow-derived macrophages were observed to migrate into the retina of Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice. These invading cells were still observed in the subretinal space in 18-week-old animals.ConclusionsCx3cr1-GFP-positive microglia and Ccr2-RFP-positive macrophages were distinguishable in the retinas of Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice. In addition, Ccr2 expression in Cx3cr1 positive microglia is a feature of microglial activation in RD. Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice enabled observation of microglial activation over time during RD and may be useful for developing inflammation-targeted treatment strategies for RD in the future.