Translation efficiency is a determinant of the magnitude of miRNA-mediated repression

Abstract MicroRNAs are well known regulators of mRNA stability and translation. However, the magnitude of both translational repression and mRNA decay induced by miRNA binding varies greatly between miRNA targets. This can be the result of cis and trans factors that affect miRNA binding or action. We set out to address this issue by studying how various mRNA characteristics affect miRNA-mediated repression. Using a dual luciferase reporter system, we systematically analyzed the ability of selected mRNA elements to modulate miRNA-mediated repression. We found that changing the 3′UTR of a miRNA-targeted reporter modulates translational repression by affecting the translation efficiency. This 3′UTR dependent modulation can be further altered by changing the codon-optimality or 5′UTR of the luciferase reporter. We observed maximal repression with intermediate codon optimality and weak repression with very high or low codon optimality. Analysis of ribosome profiling and RNA-seq data for endogenous miRNA targets revealed translation efficiency as a key determinant of the magnitude of miRNA-mediated translational repression. Messages with high translation efficiency were more robustly repressed. Together our results reveal modulation of miRNA-mediated repression by characteristics and features of the 5′UTR, CDS and 3′UTR

Urb-RIP - An adaptable and efficient approach for immunoprecipitation of RNAs and associated RNAs/proteins

Post-transcriptional regulation of gene expression is an important process that is mediated by interactions between mRNAs and RNA binding proteins (RBP), non-coding RNAs (ncRNA) or ribonucleoproteins (RNP). Key to the study of post-transcriptional regulation of mRNAs and the function of ncRNAs such as long non-coding RNAs (lncRNAs) is an understanding of what factors are interacting with these transcripts. While several techniques exist for the enrichment of a transcript whether it is an mRNA or an ncRNA, many of these techniques are cumbersome or limited in their application. Here we present a novel method for the immunoprecipitation of mRNAs and ncRNAs, Urb-RNA immunoprecipitation (Urb-RIP). This method employs the RRM1 domain of the "resurrected" snRNA-binding protein Urb to enrich messages containing a stem-loop tag. Unlike techniques which employ the MS2 protein, which require large repeats of the MS2 binding element, Urb-RIP requires only one stem-loop. This method routinely provides over ~100-fold enrichment of tagged messages. Using this technique we have shown enrichment of tagged mRNAs and lncRNAs as well as miRNAs and RNA-binding proteins bound to those messages. We have confirmed, using Urb-RIP, interaction between RNA PolIII transcribed lncRNA BC200 and polyA binding protein

PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs

A large number of RNA binding proteins (RBPs) and miRNAs bind to the 3′ untranslated regions of mRNA, but methods to dissect their function and interactions are lacking. Here the authors introduce post-transcriptional regulatory element sequencing (PTRE-seq) to dissect sequence preferences, interactions and consequences of RBP and miRNA binding

Sirtuin-mediated nuclear differentiation and programmed degradation in Tetrahymena

<p>Abstract</p> <p>Background</p> <p>The NAD⁺-dependent histone deacetylases, known as "sirtuins", participate in a variety of processes critical for single- and multi-cellular life. Recent studies have elucidated the importance of sirtuin activity in development, aging, and disease; yet, underlying mechanistic pathways are not well understood. Specific sirtuins influence chromatin structure and gene expression, but differences in their pathways as they relate to distinct chromatin functions are just beginning to emerge. To further define the range of global chromatin changes dependent on sirtuins, unique biological features of the ciliated protozoan <it>Tetrahymena thermophila </it>can be exploited. This system offers clear spatial and temporal separation of multiple whole genome restructuring events critical for the life cycle.</p> <p>Results</p> <p>Inhibition with nicotinamide revealed that sirtuin deacetylase activity in <it>Tetrahymena </it>cells promotes chromatin condensation during meiotic prophase, differentiation of heterochromatin from euchromatin during development, and chromatin condensation/degradation during programmed nuclear death. We identified a class I sirtuin, called Thd14, that resides in mitochondria and nucleoli during vegetative growth, and forms a large sub-nuclear aggregate in response to prolonged cell starvation that may be peripherally associated with nucleoli. During sexual conjugation and development Thd14 selectively concentrates in the parental nucleus prior to its apoptotic-like degradation.</p> <p>Conclusions</p> <p>Sirtuin activity is important for several functionally distinct events requiring global chromatin condensation. Our findings suggest a novel role for sirtuins in promoting programmed pycnosis by acting on chromatin destined for degradation. The sirtuin Thd14, which displays physiological-dependent differential localization within the nucleus, is a candidate for a chromatin condensation enzyme that is coupled to nuclear degradation.</p

Induction of viral mimicry upon loss of DHX9 and ADAR1 in breast cancer cells

UNLABELLED: Detection of viral double-stranded RNA (dsRNA) is an important component of innate immunity. However, many endogenous RNAs containing double-stranded regions can be misrecognized and activate innate immunity. The IFN-inducible ADAR1-p150 suppresses dsRNA sensing, an essential function for adenosine deaminase acting on RNA 1 (ADAR1) in many cancers, including breast. Although ADAR1-p150 has been well established in this role, the functions of the constitutively expressed ADAR1-p110 isoform are less understood. We used proximity labeling to identify putative ADAR1-p110-interacting proteins in breast cancer cell lines. Of the proteins identified, the RNA helicase DHX9 was of particular interest. Knockdown of DHX9 in ADAR1-dependent cell lines caused cell death and activation of the dsRNA sensor PKR. In ADAR1-independent cell lines, combined knockdown of DHX9 and ADAR1, but neither alone, caused activation of multiple dsRNA sensing pathways leading to a viral mimicry phenotype. Together, these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways. SIGNIFICANCE: These findings implicate DHX9 as a suppressor of dsRNA sensing. In some cell lines, loss of DHX9 alone is sufficient to cause activation of dsRNA sensing pathways, while in other cell lines DHX9 functions redundantly with ADAR1 to suppress pathway activation

Urb-RIP enriches for mCherry-mRNA and bound PABP.

<p><b>a</b> Schematic describing 2HA-Urb construct used for Urb-RIP and reporter, mCherry (mCh)-mRNA tagged with SLII, used to validate and optimize Urb-RIP. <b>b</b> Enrichment of mCh-mRNA by Urb-RIP as determined by qPCR. The cell line 293-2HA-Urb was transfected with a plasmid expressing mCherry-mRNA untagged or tagged with SLII. Two days after transfection the cells were UV-irradiated at 400 mJ/cm² and subsequently lysed. Immunoprecipitation was performed using the Urb-RIP protocol and RNA was eluted with proteinase K treatment. qRT-PCR was performed using mCh and GAPDH primers. <b>c</b> Comparison of relative expression amounts of mCherry with (+SLII) and without (-SLII) tagging. qPCR results show a modest reduction in mCherry expression upon insertion of SLII. Relative levels of mCherry are normalized to GAPDH. <b>d</b> western blot shows enrichment of PABP following Urb-RIP of mCh. Half of the immunoprecipitate from above was eluted with sample buffer and analyzed by western blot with antibody against proteins listed. Samples labeled input represent 5% of the total sample used for Urb-RIP. <b>e</b> quantification of western blot in <b>d</b>. For panels <b>b</b> and <b>c</b> mean ± SD of three independent experiments are shown. For panel <b>e</b> mean ±SD of three independent experiments are shown. * p<0.05, ** p<0.01.</p

Schematic illustration of the Urb-RIP protocol and potential applications.

<p>The first step of the Urb-RIP protocol is to tag an RNA of interest with the SLII-tag, illustrated in the figure. The tagged RNA and in parallel an untagged control are coexpressed with 2HA-Urb in a cell line of interest. After a period of time the cells are UV irradiated to produce RNA-protein crosslinks. The cells are then lysed and immunoprecipitation is performed using blocked anti-HA magnetic beads. The RNA or protein is then eluted and analyzed by an appropriate method. This approach should be applicable to mRNAs and lncRNAs and amendable to a wide range of methods for eluate analysis.</p

Urb-RIP shows Argonaute and miRNA binding to miRNA-targeted messages in human cells.

<p><b>a</b> Schematic describing the <i>let-7</i> reporters used to validate Urb-RIPs ability to identify interacting miRNA. <b>b</b> Enrichment of <i>let-7</i> and mCh-mRNA by Urb-RIP as determined by qPCR. The cell line 293-2HA-Urb was transfected with plasmids expressing the constructs described in <b>a</b> as well as a plasmid for expression of GFP-FLAG-Ago2. Two days after transfection the cells were UV-irradiated at 400 mJ/cm² and subsequently lysed. Immunoprecipitation was performed using the Urb-RIP protocol and RNA was eluted with proteinase K treatment. qRT-PCR was performed using mCh, <i>let-7</i> and GAPDH primers. <b>c</b> enrichment of <i>let-7</i> normalized to mCh abundance in the immunoprecipitate. <b>d</b> western blot shows enrichment of GFP-FLAG-Ago2 following Urb-RIP. The mCh-<i>let-7</i>-SLII reporters from above were co-transfected with GFP-FLAG-Ago2. Two days after transfection Urb-RIP was performed. The eluted protein as well as input was analyzed by western blot with antibody against FLAG (GFP-FLAG-Ago2 and 2HA-FLAG-Urb). Samples labeled input represent 5% of the total sample used for Urb-RIP. <b>e</b> quantification of western blot in <b>d,</b> normalized to 2HA-Urb, relative to mCh-<i>let-7</i>-SLII.</p