A key prerequisite to understand how gene regulatory processes are controlled by the interplay of RNA-binding proteins and ribonucleoprotein complexes with RNAs is the generation of comprehensive ...high-resolution maps of protein–RNA interactions. Recent advances in next-generation sequencing technology accelerated the development of various crosslinking and immunoprecipitation (CLIP) approaches to broadly identify RNA regions contacted by RNA-binding proteins. However these methods only consider single RNA-binding proteins and their contact sites, irrespective of the overall cis-regulatory sequence space contacted by other RNA interacting factors. Here we describe the application of protein occupancy profiling, a novel approach that globally displays the RNA contact sites of the poly(A)+ RNA-bound proteome. Protein occupancy profiling enables the generation of transcriptome-wide maps of protein–RNA interactions on polyadenylated transcripts and narrows the sequence search space for transcript regions involved in cis-regulation of gene expression in response to internal or external stimuli, altered cellular programs or disease.
Both sickle cell disease and β-thalassemia are major sources of morbidity and mortality worldwide. Continued production of the β-like γ-globin genes that form fetal hemoglobin after infancy has been ...shown to ameliorate the severity of these disorders. As a result, there has been considerable interest in understanding the underlying regulation of the physiologic fetal-to-adult hemoglobin switch in humans to be able to better manipulate this process for therapeutic purposes. To date, only a single factor, BCL11A, has been identified as being involved in the developmental regulation of human hemoglobin switching. BCL11A is a direct transcriptional repressor of the γ-globin genes. Moreover, BCL11A is expressed in a developmental stage-specific manner to regulate human hemoglobin switching. However, despite extensive studies, the mechanisms that act upstream to regulate BCL11A expression and thereby control hemoglobin switching have remained elusive. To gain further insights, we have directly explored the developmental regulation of BCL11A expression at various stages of human erythropoiesis. We find that BCL11A is regulated at the level of mRNA translation during development. While BCL11A mRNA is comparably expressed in a similar manner at all developmental stages in erythroid cells, robust protein expression only occurs in adult erythroid cells. Importantly, we demonstrate that at the earlier stages of development, the observed reduction in protein expression is attributable to decreased synthesis and not increased degradation of BCL11A through direct assessment of both protein synthesis and degradation rates in primary erythroid cells from various stages of human development. Interestingly, while BCL11A protein is not well synthesized at these earlier stages of development, we find that its mRNA curiously continues to be associated with ribosomes in a comparable manner between newborn and adult erythroid precursors using polysome fractionation of stage-matched erythroid cells. Through use of an unbiased proteomic analysis approach involving RNA affinity purification of the 18S ribosomal RNA in erythroid cells, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a reciprocal pattern to BCL11A, directly interacts with ribosomes. We additionally show that the observed suppression of BCL11A protein translation is mediated by LIN28B. Using a newly developed RNA isolation and crosslinking immunoprecipitation approach coupled to massively parallel sequencing we mapped the direct interaction of LIN28B with BCL11A mRNA at nucleotide resolution. Through a number of functional assays in both newborn and adult erythroid cells, we additionally demonstrate that this alteration of BCL11A translation is entirely independent of the role of LIN28B in the biogenesis of let-7 microRNAs. Finally, we show that BCL11A is the major functional target in LIN28B-mediated fetal hemoglobin induction through functional complementation experiments in primary human erythroid cells. Our results reveal a previously unappreciated regulatory mechanism underlying human hemoglobin switching. Moreover, our findings highlight opportunities for developing improved treatments for sickle cell disease and β-thalassemia by understanding the upstream regulation of human hemoglobin switching.
No relevant conflicts of interest to declare.
Fragile-X Syndrome (FXS) is a multi-organ disease leading to mental retardation, macro-orchidism in males, and premature ovarian insufficiency in female carriers. FXS is also a prominent monogenic ...disease associated with autism spectrum disorders (ASD). FXS is typically caused by the loss of
FRAGILE X-MENTAL RETARDATION 1
(
FMR1
) expression, which encodes for the RNA-binding protein (RBP), FMRP. We report the discovery of distinct RNA recognition elements (RREs) that correspond to the two independent RNA binding domains of FMRP, and the binding sites within the mRNA targets for wild-type and I304N mutant FMRP isoforms and its paralogs, FXR1 and FXR2. RRE frequency, ratio, and distribution determine target mRNA association with FMRP. Among highly-enriched targets, we identified many genes involved in ASD and demonstrate that FMRP affects their protein levels in cell culture, mice, and human brain. Unexpectedly, we discovered that these targets are also dysregulated in
Fmr1
-/-
mouse ovaries, showing signs of premature follicular overdevelopment. These results indicate that FMRP targets shared signaling pathways across different cellular contexts. As it is become increasingly appreciated that signaling pathways are important to FXS and ASD, our results here provide a molecular guide towards the pursuit of novel therapeutic targets for these neurological disorders.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Circular RNAs (circRNAs) in animals are an enigmatic class of RNA with unknown function. To explore circRNAs systematically, we sequenced and computationally analysed human, mouse and nematode RNA. ...We detected thousands of well-expressed, stable circRNAs, often showing tissue/developmental-stage-specific expression. Sequence analysis indicated important regulatory functions for circRNAs. We found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 63 conserved binding sites for the ancient miRNA miR-7. Further analyses indicated that CDR1as functions to bind miR-7 in neuronal tissues. Human CDR1as expression in zebrafish impaired midbrain development, similar to knocking down miR-7, suggesting that CDR1as is a miRNA antagonist with a miRNA-binding capacity ten times higher than any other known transcript. Together, our data provide evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to-tail splicing of exons, suggesting previously unrecognized regulatory potential of coding sequences.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background and Purpose — GV150526 , a selective glycine site antagonist, reduces infarct volume in rats with focal cerebral ischemia. Safety and efficacy in humans with acute stroke are being ...investigated. We sought to further explore the safety, pharmacokinetics, and preliminary outcome of GV150526 treatment in patients with a clinical diagnosis of acute stroke. Methods —Two trials were conducted in North America. The North American Glycine Antagonist in Neuroprotection trial (GAIN 1) (GLYA2001; United States only) was designed as a sequential dose escalation study. GAIN 2 (GLYA2005; United States and Canada) was designed to further assess the safety of the highest dose tolerated in GAIN 1. Both trials were randomized (2:1), double-blind, and placebo controlled. Treatment was started within 12 hours of symptom onset; patients with both ischemic stroke and primary intracerebral hemorrhage were included in both trials. Results —The dose escalation study (GAIN 1) completed 3 dosing tiers. Enrollment was suspended before escalation to the fourth tier because of laboratory reports of transiently elevated bilirubin levels in a concurrent European study that employed the dose targeted for this tier. After review by an independent safety committee of the worldwide safety data, the second study (GAIN 2) commenced. One hundred nine patients were randomized and dosed with study drug, either an 800-mg loading dose followed by 200 mg every 12 hours for 3 days of GV150526 or placebo. The incidence of serious adverse events was similar in the drug and placebo groups. Mild irritation at the infusion site and symptoms suggestive of mild and reversible altered mentation were reported more frequently in the GV150526 group than in the placebo group. Hyperbilirubinemia was reported in 6% of GV150526 -treated patients compared with 3% of placebo-treated patients. Outcome at 4 weeks after stroke was better in GV150526 -treated patients, but the studies were not powered to show statistical significance, and the baseline neurological deficits in the GV150526 -treated patients were less severe. Conclusions —These preliminary studies suggest that GV150526 is well tolerated by patients with suspected acute stroke. Further pivotal studies testing the efficacy and safety of GV150526 in acute stroke are ongoing.
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