mRNA Fragments Research Articles

Secretion of Mutant DNA and mRNA by the Exosomes of Breast Cancer Cells.

Exosomes are the small vesicles that are secreted by different types of normal and tumour cells and can incorporate and transfer their cargo to the recipient cells. The main goal of the present work was to study the tumour exosomes’ ability to accumulate the parent mutant DNA or RNA transcripts with their following transfer to the surrounding cells. The experiments were performed on the MCF7 breast cancer cells that are characterized by the unique coding mutation in the PIK3CA gene. Using two independent methods, Sanger sequencing and allele-specific real-time PCR, we revealed the presence of the fragments of the mutant DNA and RNA transcripts in the exosomes secreted by the MCF7 cells. Furthermore, we demonstrated the MCF7 exosomes’ ability to incorporate into the heterologous MDA-MB-231 breast cancer cells supporting the possible transferring of the exosomal cargo into the recipient cells. Sanger sequencing of the DNA from MDA-MB-231 cells (originally bearing a wild type of PIK3CA) treated with MCF7 exosomes showed no detectable amount of mutant DNA or RNA; however, using allele-specific real-time PCR, we revealed a minor signal from amplification of a mutant allele, showing a slight increase of mutant DNA in the exosome-treated MDA-MB-231 cells. The results demonstrate the exosome-mediated secretion of the fragments of mutant DNA and mRNA by the cancer cells and the exosomes’ ability to transfer their cargo into the heterologous cells.

Estimation of peptide elongation times from ribosome profiling spectra.

Ribosome profiling spectra bear rich information on translation control and dynamics. Yet, due to technical biases in library generation, extracting quantitative measures of discrete translation events has remained elusive. Using maximum likelihood statistics and data set from Escherichia coli we develop a robust method for neutralizing technical biases (e.g. base specific RNase preferences in ribosome-protected mRNA fragments (RPF) generation), which allows for correct estimation of translation times at single codon resolution. Furthermore, we validated the method with available datasets from E. coli treated with antibiotic to inhibit isoleucyl-tRNA synthetase, and two datasets from Saccharomyces cerevisiae treated with two RNases with distinct cleavage signatures. We demonstrate that our approach accounts for RNase cleavage preferences and provides bias-corrected translation times estimates. Our approach provides a solution to the long-standing problem of extracting reliable information about peptide elongation times from highly noisy and technically biased ribosome profiling spectra.

Impact of Ribosome Activity on SARS-CoV-2 LNP - Based mRNA Vaccines.

Coronavirus-related Severe Acute Respiratory Syndrome-2 (SARS-CoV-2) initially was detected in Wuhan, Hubei, China. Since early 2021, World Health Organization (WHO) has declared Coronavirus Disease 2019 (COVID-19) a pandemic due to rapidly transformed to a globally massive catastrophic viral infection. In order to confront this emergency situation, many pharmaceutical companies focused on the design and development of efficient vaccines that are considered necessary for providing a level of normalization in totally affected human social-economical activity worldwide. A variety of vaccine types are under development, validation or even some of them have already completed these stages, initially approved as conditional marketing authorisation by Food and Drug Administration (FDA), European Medicines Agency (EMA), and other national health authorities for commercial purposes (in vivo use in general population), accelerating their production and distribution process. Innovative nucleoside-modified viral messenger RNA (v-mRNA)—based vaccines encapsulated within nanoparticles—specifically lipid ones (LNPs)—are now well recognized. Although this is a promising genetic engineering topic in the field of nanopharmacogenomics or targeted nucleic vaccines, there are limited but continuously enriched in vivo data in depth of time regarding their safety, efficacy, and immune response. In the current paper we expand the limited published data in the field of ribosome machinery and SARS-CoV-2 mRNA fragment vaccines interaction by describing their functional specialization and modifications. Additionally, alterations in post-transcriptional/translational molecules and mechanisms that could potentially affect the interaction between target cells and vaccines are also presented. Understanding these mechanisms is a crucial step for the next generation v-mRNA vaccines development.

Circulating PTHrP measurement in the assessment of severe hypercalcaemia in an infant

Parathyroid hormone-related protein (PTHrP) can simulate most of the actions of parathyroid hormone (PTH) including increases in bone resorption and distal tubular calcium reabsorption and inhibition of proximal tubular phosphate...

RiboA: a web application to identify ribosome A-site locations in ribosome profiling data

BackgroundTranslation is a fundamental process in gene expression. Ribosome profiling is a method that enables the study of transcriptome-wide translation. A fundamental, technical challenge in analyzing Ribo-Seq data is identifying the A-site location on ribosome-protected mRNA fragments. Identification of the A-site is essential as it is at this location on the ribosome where a codon is translated into an amino acid. Incorrect assignment of a read to the A-site can lead to lower signal-to-noise ratio and loss of correlations necessary to understand the molecular factors influencing translation. Therefore, an easy-to-use and accurate analysis tool is needed to accurately identify the A-site locations.ResultsWe present RiboA, a web application that identifies the most accurate A-site location on a ribosome-protected mRNA fragment and generates the A-site read density profiles. It uses an Integer Programming method that reflects the biological fact that the A-site of actively translating ribosomes is generally located between the second codon and stop codon of a transcript, and utilizes a wide range of mRNA fragment sizes in and around the coding sequence (CDS). The web application is containerized with Docker, and it can be easily ported across platforms.ConclusionsThe Integer Programming method that RiboA utilizes is the most accurate in identifying the A-site on Ribo-Seq mRNA fragments compared to other methods. RiboA makes it easier for the community to use this method via a user-friendly and portable web application. In addition, RiboA supports reproducible analyses by tracking all the input datasets and parameters, and it provides enhanced visualization to facilitate scientific exploration. RiboA is available as a web service at https://a-site.vmhost.psu.edu/. The code is publicly available at https://github.com/obrien-lab/aip_web_docker under the MIT license.

RP-REP Ribosomal Profiling Reports: an open-source cloud-enabled framework for reproducible ribosomal profiling data processing, analysis, and result reporting

Ribosomal profiling is an emerging experimental technology to measure protein synthesis by sequencing short mRNA fragments undergoing translation in ribosomes. Applied on the genome wide scale, this is a powerful tool to profile global protein synthesis within cell populations of interest. Such information can be utilized for biomarker discovery and detection of treatment-responsive genes. However, analysis of ribosomal profiling data requires careful preprocessing to reduce the impact of artifacts and dedicated statistical methods for visualizing and modeling the high-dimensional discrete read count data. Here we present Ribosomal Profiling Reports (RP-REP), a new open-source cloud-enabled software that allows users to execute start-to-end gene-level ribosomal profiling and RNA-Seq analysis on a pre-configured Amazon Virtual Machine Image (AMI) hosted on AWS or on the user’s own Ubuntu Linux server. The software works with FASTQ files stored locally, on AWS S3, or at the Sequence Read Archive (SRA). RP-REP automatically executes a series of customizable steps including filtering of contaminant RNA, enrichment of true ribosomal footprints, reference alignment and gene translation quantification, gene body coverage, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially translated genes, and generation of heatmaps, co-translated gene clusters, enriched pathways, and other custom visualizations. RP-REP provides functionality to contrast RNA-SEQ and ribosomal profiling results, and calculates translational efficiency per gene. The software outputs a PDF report and publication-ready table and figure files. As a use case, we provide RP-REP results for a dengue virus study that tested cytosol and endoplasmic reticulum cellular fractions of human Huh7 cells pre-infection and at 6 h, 12 h, 24 h, and 40 h post-infection. Case study results, Ubuntu installation scripts, and the most recent RP-REP source code are accessible at GitHub. The cloud-ready AMI is available at AWS (AMI ID: RPREP RSEQREP (Ribosome Profiling and RNA-Seq Reports) v2.1 (ami-00b92f52d763145d3)).

RP-REP Ribosomal Profiling Reports: an open-source cloud-enabled framework for reproducible ribosomal profiling data processing, analysis, and result reporting.

Ribosomal profiling is an emerging experimental technology to measure protein synthesis by sequencing short mRNA fragments undergoing translation in ribosomes. Applied on the genome wide scale, this is a powerful tool to profile global protein synthesis within cell populations of interest. Such information can be utilized for biomarker discovery and detection of treatment-responsive genes. However, analysis of ribosomal profiling data requires careful preprocessing to reduce the impact of artifacts and dedicated statistical methods for visualizing and modeling the high-dimensional discrete read count data. Here we present Ribosomal Profiling Reports (RP-REP), a new open-source cloud-enabled software that allows users to execute start-to-end gene-level ribosomal profiling and RNA-Seq analysis on a pre-configured Amazon Virtual Machine Image (AMI) hosted on AWS or on the user's own Ubuntu Linux server. The software works with FASTQ files stored locally, on AWS S3, or at the Sequence Read Archive (SRA). RP-REP automatically executes a series of customizable steps including filtering of contaminant RNA, enrichment of true ribosomal footprints, reference alignment and gene translation quantification, gene body coverage, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially translated genes, and generation of heatmaps, co-translated gene clusters, enriched pathways, and other custom visualizations. RP-REP provides functionality to contrast RNA-SEQ and ribosomal profiling results, and calculates translational efficiency per gene. The software outputs a PDF report and publication-ready table and figure files. As a use case, we provide RP-REP results for a dengue virus study that tested cytosol and endoplasmic reticulum cellular fractions of human Huh7 cells pre-infection and at 6h, 12h, 24h, and 40h post-infection. Case study results, Ubuntu installation scripts, and the most recent RP-REP source code are accessible at GitHub. The cloud-ready AMI is available at AWS (AMI ID: RPREP RSEQREP (Ribosome Profiling and RNA-Seq Reports) v2.1 (ami-00b92f52d763145d3)).

Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection.

Short, structured fragments of non-coding mRNA may act as molecular switches upon binding specific ligands, regulating the translation of proteins encoded downstream this mRNA sequence. One switch, called riboswitch N1, is regulated by aminoglycosides such as neomycin. Nucleobase mutations in the apical loop, although distant from the binding pocket, significantly affect neomycin affinity and riboswitch regulatory efficiency. To explain this influence, we conducted molecular dynamics simulations using generalized replica exchange with solute tempering (gREST). Translation assay of a reporter protein in a yeast system shows that mutating A17 to G in the riboswitch apical loop reduces 6-fold the translation regulation efficiency of the mutant. Indeed, simulations of the unbound riboswitch show that G17 frequently stacks with base 7, while base 8 is stabilized towards the binding site in a way that it may interfere with the conformational selection mechanism and decrease riboswitch regulatory activity. In the riboswitch complexes, this single-point A to G mutation disrupts a strong hydrogen bond between nucleotides 5 and 17 and, instead, a new hydrogen bond between residue 17 and neomycin is created. This change forces neomycin to occupy a slightly shifted position in the binding pocket, which increases neomycin flexibility. Our simulations of the U14C mutation suggest that the riboswitch complex with neomycin is more stable if cytosine 14 is protonated. A hydrogen bond between the RNA phosphate and protonated cytosine appears as the stabilizing factor. Also, based on the cell-free translation assay and isothermal titration calorimetry experiments, mutations of nucleotides 14 and 15 affect only slightly the riboswitch ability to bind the ligand and its activity. Indeed, the simulation of the unbound U15A mutant suggests conformations preformed for ligand binding, which may explain slightly higher regulatory activity of this mutant. Overall, our results corroborate the in vivo and in vitro experiments on the N1 riboswitch-neomycin system, detail the relationship between nucleobase mutations and RNA dynamics, and reveal the conformations playing the major role in the conformational selection mechanism.

Recognition of non-CpG repeats in Alu and ribosomal RNAs by the Z-RNA binding domain of ADAR1 induces A-Z junctions

Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted — and in some cases even shown — to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, we use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Our results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighboring A-form regions which is similar in character to what has been observed for B-Z-DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows us to propose a model for how Zα could influence the RNA binding specificity of ADAR1.

PiRNAs Coordinate Poly(UG) Tailing to Prevent Aberrant and Permanent Gene Silencing

Noncoding RNAs have emerged as mediators of transgenerational epigenetic inheritance (TEI) in a number of organisms. A robust example of RNA-directed TEI is the inheritance of gene silencing states following RNA interference (RNAi) in the metazoan C. elegans. During RNAi inheritance, gene silencing is transmitted by a self-perpetuating cascade of siRNA-directed poly(UG) tailing of mRNA fragments (pUGylation), followed by siRNA synthesis from poly(UG)-tailed mRNA templates (termed pUG RNA/siRNA cycling). Despite the self-perpetuating nature of pUG RNA/siRNA cycling, RNAi inheritance is finite, suggesting that systems likely exist to prevent permanent RNAi-triggered gene silencing. Here we show that, in the absence of Piwi-interacting RNAs (piRNAs), an animal-specific class of small noncoding RNA, RNAi-based gene silencing can become essentially permanent, lasting at near 100% penetrance for more than five years and hundreds of generations. This permanent gene silencing is mediated by perpetual activation of the pUG RNA/siRNA TEI pathway. Further, we find that piRNAs coordinate endogenous RNAi pathways to prevent germline-expressed genes, which are not normally subjected to TEI, from entering a state of permanent and irreversible epigenetic silencing also mediated by perpetual activation of pUG RNA/siRNA cycling. Together, our results show that one function of C. elegans piRNAs is to insulate germline-expressed genes from aberrant and runaway inactivation by the pUG RNA/siRNA epigenetic inheritance system.

H3K9ac modification was involved in doxorubicin induced apoptosis by regulating Pik3ca transcription in H9C2 cells

AimWe evaluated the effect of H3K9ac modification on the transcriptional level of Pik3ca and the apoptosis induction effects of Pik3ca in the PI3K/AKT pathway in rat H9C2 cells. Main methodsH9C2 cells were cultured with 1uM doxorubicin for 8 h. The acetylation level of histone H3K9 in the transcriptional initiation area of Pik3ca was determined by CHIP-seq. The enrichment of mRNA fragment of Pik3ca transcription initiation region by H3K9ac antibody was detected by CHIP-qPCR, and the expression of Pik3ca was detected by real-time Polymerase Chain Reaction(rt-PCR) and western blot. The transcription efficiency of Pik3ca siRNA was detected by immunofluorescence and western blot. Cell Counting Kit8(CCK8) was used to detect the cell activity and flow cytometry was used to detect the apoptosis rate. Western blot was applied to assess the protein expression level of PI3K, P-AKT, AKT, Bcl2, BAX and cleaved-caspase3 in H9C2 cells. Key findingsIn doxorubicin-inducedH9C2 cells, the acetylation levels of histone H3K9 in the Pik3ca transcriptional initiation region significantly increased and promoted the transcription of Pik3ca. After knockdown of Pik3ca, the PI3K/AKT signaling pathway was inhibited, and the protein expression of Bcl2 was increased, the protein expression of BAX and cleaved-caspase3 were decreased. PI3K/AKT pathway activator partially reversed the effect of silencing Pik3ca. SignificanceIn summary, the elevated H3K9ac level in the Pik3ca transcriptional initiation region promoted the transcription of Pik3ca, and Pik3ca promoted doxorubicin induced apoptosis in H9C2 cells by activating the PI3K/AKT pathway, providing a new theoretical basis for the study of doxorubicin cardiomyopathy.

Disome-seq reveals widespread ribosome collisions that promote cotranslational protein folding

BackgroundThe folding of proteins is challenging in the highly crowded and sticky environment of a cell. Regulation of translation elongation may play a crucial role in ensuring the correct folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes by ribo-seq. However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation.ResultsIn this study, we performed disome-seq to sequence mRNA fragments protected by two stacked ribosomes, a product of translational pauses during which the 5′-elongating ribosome collides with the 3′-paused one. We detected widespread ribosome collisions that are related to slow ribosome release when stop codons are at the A-site, slow peptide bond formation from proline, glycine, asparagine, and cysteine when they are at the P-site, and slow leaving of polylysine from the exit tunnel of ribosomes. The structure of disomes obtained by cryo-electron microscopy suggests a different conformation from the substrate of the ribosome-associated protein quality control pathway. Collisions occurred more frequently in the gap regions between α-helices, where a translational pause can prevent the folding interference from the downstream peptides. Paused or collided ribosomes are associated with specific chaperones, which can aid in the cotranslational folding of the nascent peptides.ConclusionsTherefore, cells use regulated ribosome collisions to ensure protein homeostasis.

Comprehensive profiling of mRNA polyadenylation in specific cell types in vivo by cTag-PAPERCLIP.

The ability to generate cell-type specific mRNA polyadenylation (pA) maps from complex tissues is crucial for understanding how alternative polyadenylation (APA) is regulated in individual cell types in their physiological environment under different conditions. In this chapter, we discuss cTag-PAPERCLIP, a recently developed method combining the well-established CLIP (crosslinking immunoprecipitation) technique and the Cre-lox system to achieve customized cell-type specific APA profiling from mouse tissue without cell purification or enrichment. In cTag-PAPERCLIP, selective expression of GFP-tagged poly(A) binding protein (PABP-GFP) in the desired cell type is achieved through Cre-mediated activation of a latent knock-in allele of PABP-GFP. Immunoprecipitation of PABP-GFP then allows mRNA 3' end fragments in the desired cell type to be specifically retrieved from ultraviolet (UV)-irradiated whole tissue lysate. The mRNA fragments are subsequently turned into a cDNA library to provide a comprehensive APA map and an mRNA expression profile of the chosen cell type through deep sequencing.

Analysis of Stored mRNA Degradation in Acceleratedly Aged Seeds of Wheat and Canola in Comparison to Arabidopsis.

Seed aging has become a topic of renewed interest but its mechanism remains poorly understood. Our recent analysis of stored mRNA degradation in aged Arabidopsis seeds found that the stored mRNA degradation rates (estimated as the frequency of breakdown per nucleotide per day or β value) were constant over aging time under stable conditions. However, little is known about the generality of this finding to other plant species. We expanded the analysis to aged seeds of wheat (Triticum aestivum) and canola (Brassica napus). It was found that wheat and canola seeds required much longer periods than Arabidopsis seeds to lose seed germination ability completely under the same aging conditions. As what had been observed for Arabidopsis, stored mRNA degradation (∆Ct value in qPCR) in wheat and canola seeds correlated linearly and tightly with seed aging time or mRNA fragment size, while the quality of total RNA showed little change during seed aging. The generated β values reflecting the rate of stored mRNA degradation in wheat or canola seeds were similar for different stored mRNAs assayed and constant over seed aging time. The overall β values for aged seeds of wheat and canola showed non-significant differences from that of Arabidopsis when aged under the same conditions. These results are significant, allowing for better understanding of controlled seed aging for different species at the molecular level and for exploring the potential of stored mRNAs as seed aging biomarkers.

A simple and sensitive direct mRNA multiplexed detection strategy for amoA-targeted monitoring of ammonia-oxidizing activity in water environment

Nitrogenous pollutants are the main causes of water eutrophication, which greatly interfere with the balance of the aquatic ecosystem. The biological nitrogen removal using bacteria is the leading technology to control nitrogenous pollutants. The α-subunit (amoA) of ammonia monooxygenase (AMO) of aerobic ammonia-oxidizing bacteria (AOB) is an ammonia-depleting biomarker in nitrogen removal processes. Currently, RT-PCR and metatranscriptome are the general methods for analyzing amoA gene expression, however they can introduce amplification bias and are time consuming. Herein we report a new direct mRNA multiplexed detection strategy based on xMAP array, for analyzing gene expression without reverse transcription and amplification steps. After analyzing all amoA sequences in database, we design a universal probe for AOB with coverage of 79.8% whose high coverage is further confirmed by metatranscriptome. Moreover, we classify AOB into 10 clusters, and then design the specific probes of each cluster. This technique enables single-nucleotide, wide linear dynamic range (5–50,000 amol) and high sensitivity (5 amol with detection volume of 1 µL) detection with using locked-nucleic-acid probes. Particularly, the sensitivity is by far among the highest level for multiplex mRNA detection without amplification. We found that mRNA fragmentation is not necessary in our direct detection, simplifying our strategy. We have successfully detected amoA mRNA semi-quantitatively and determined the predominant cluster in sludge from wastewater treatment plants, which was consistent with RT-PCR and metatranscriptome results. Meanwhile, the ammonia-oxidizing activities of sludge verified the amoA mRNA detection results. In conclusion, we have developed a mRNA direct, simple, specific and sensitive multiplexed detection strategy, which shows great potential for application in gene expression analysis, environmental microbial detection and activity diagnosis of wastewater treatment plants.

Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense Oligoribonucleotide.

Antisense DNA oligonucleotides, short interfering RNAs (siRNAs), and CRISPR/Cas9 genetic tools are the most useful therapeutic nucleic acids regulating gene expression based on the antisense specificity towards messenger RNA. Here, we present an effective novel strategy for inhibiting translation based on the antisense-controlled formation of an RNA quadruplex-duplex hybrid (QDH) between a G-rich RNA antisense oligoribonucleotide (Q-ASO) and specific mRNA, comprising two distant G-tracts. We selected epidermal growth factor receptor (EGFR) as a well-established target protein in anticancer therapy. The chemically modified, bi-functional anti-EGFR Q-ASO and a 56-nt long EGFR mRNA fragment, in the presence of potassium ions, were shown to form in vitro very stable parallel G-quadruplex containing a 28-nt long external loop folding to two duplex-stem structure. Besides, the Q-ASOs effectively reduced EGFR mRNA levels compared to the non-modified RNA and DNA antisense oligonucleotides (rASO, dASO). In addition, the hybridization specificity of Q-ASO comprising a covalently attached fluorescent tag was confirmed in living cells by visualization of the G4 green fluorescent species in the presence of other antisense inhibitors under competitive conditions. The results presented here offer novel insights into the potential application of Q-ASOs for the detection and/or alteration of (patho)biological processes through RNA:RNA quadruplex-duplex formation in cellular systems.

Group II Introns Generate Functional Chimeric Relaxase Enzymes with Modified Specificities through Exon Shuffling at Both the RNA and DNA Level.

Group II introns are large self-splicing RNA enzymes with a broad but somewhat irregular phylogenetic distribution. These ancient retromobile elements are the proposed ancestors of approximately half the human genome, including the abundant spliceosomal introns and non-long terminal repeat retrotransposons. In contrast to their eukaryotic derivatives, bacterial group II introns have largely been considered as harmful selfish mobile retroelements that parasitize the genome of their host. As a challenge to this view, we recently uncovered a new intergenic trans-splicing pathway that generates an assortment of mRNA chimeras. The ability of group II introns to combine disparate mRNA fragments was proposed to increase the genetic diversity of the bacterial host by shuffling coding sequences. Here, we show that the Ll.LtrB and Ef.PcfG group II introns from Lactococcus lactis and Enterococcus faecalis respectively can both use the intergenic trans-splicing pathway to catalyze the formation of chimeric relaxase mRNAs and functional proteins. We demonstrated that some of these compound relaxase enzymes yield gain-of-function phenotypes, being significantly more efficient than their precursor wild-type enzymes at supporting bacterial conjugation. We also found that relaxase enzymes with shuffled functional domains are produced in biologically relevant settings under natural expression levels. Finally, we uncovered examples of lactococcal chimeric relaxase genes with junctions exactly at the intron insertion site. Overall, our work demonstrates that the genetic diversity generated by group II introns, at the RNA level by intergenic trans-splicing and at the DNA level by recombination, can yield new functional enzymes with shuffled exons, which can lead to gain-of-function phenotypes.

TAF1 Transcripts and Neurofilament Light Chain as Biomarkers for X-linked Dystonia-Parkinsonism.

ABSTRACTBackgroundX‐linked dystonia‐parkinsonism is a rare neurological disease endemic to the Philippines. Dystonic symptoms appear in males at the mean age of 40 years and progress to parkinsonism with degenerative pathology in the striatum. A retrotransposon inserted in intron 32 of the TAF1 gene leads to alternative splicing in the region and a reduction of the full‐length mRNA transcript.ObjectivesThe objective of this study was to discover cell‐based and biofluid‐based biomarkers for X‐linked dystonia‐parkinsonism.MethodsRNA from patient‐derived neural progenitor cells and their secreted extracellular vesicles were used to screen for dysregulation of TAF1 expression. Droplet‐digital polymerase chain reaction was used to quantify the expression of TAF1 mRNA fragments 5′ and 3′ to the retrotransposon insertion and the disease‐specific splice variant TAF1‐32i in whole‐blood RNA. Plasma levels of neurofilament light chain were measured using single‐molecule array.ResultsIn neural progenitor cells and their extracellular vesicles, we confirmed that the TAF1‐3′/5′ ratio was lower in patient samples, whereas TAF1‐32i expression is higher relative to controls. In whole‐blood RNA, both TAF1‐3′/5′ ratio and TAF1‐32i expression can differentiate patient (n = 44) from control samples (n = 18) with high accuracy. Neurofilament light chain plasma levels were significantly elevated in patients (n = 43) compared with both carriers (n = 16) and controls (n = 21), with area under the curve of 0.79.Conclusions TAF1 dysregulation in blood serves as a disease‐specific biomarker that could be used as a readout for monitoring therapies targeting TAF1 splicing. Neurofilament light chain could be used in monitoring neurodegeneration and disease progression in patients. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Identification of protein-protected mRNA fragments and structured excised intron RNAs in human plasma by TGIRT-seq peak calling.

Human plasma contains > 40,000 different coding and non-coding RNAs that are potential biomarkers for human diseases. Here, we used thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) combined with peak calling to simultaneously profile all RNA biotypes in apheresis-prepared human plasma pooled from healthy individuals. Extending previous TGIRT-seq analysis, we found that human plasma contains largely fragmented mRNAs from > 19,000 protein-coding genes, abundant full-length, mature tRNAs and other structured small non-coding RNAs, and less abundant tRNA fragments and mature and pre-miRNAs. Many of the mRNA fragments identified by peak calling correspond to annotated protein-binding sites and/or have stable predicted secondary structures that could afford protection from plasma nucleases. Peak calling also identified novel repeat RNAs, miRNA-sized RNAs, and putatively structured intron RNAs of potential biological, evolutionary, and biomarker significance, including a family of full-length excised intron RNAs, subsets of which correspond to mirtron pre-miRNAs or agotrons.

Molecular basis of the selective processing of short mRNA substrates by the DcpS mRNA decapping enzyme

The 5' messenger RNA (mRNA) cap structure enhances translation and protects the transcript against exonucleolytic degradation. During mRNA turnover, this cap is removed from the mRNA. This decapping step is catalyzed by the Scavenger Decapping Enzyme (DcpS), in case the mRNA has been exonucleolyticly shortened from the 3' end by the exosome complex. Here, we show that DcpS only processes mRNA fragments that are shorter than three nucleotides in length. Based on a combination of methyl transverse relaxation optimized (TROSY) NMR spectroscopy and X-ray crystallography, we established that the DcpS substrate length-sensing mechanism is based on steric clashes between the enzyme and the third nucleotide of a capped mRNA. For longer mRNA substrates, these clashes prevent conformational changes in DcpS that are required for the formation of a catalytically competent active site. Point mutations that enlarge the space for the third nucleotide in the mRNA body enhance the activity of DcpS on longer mRNA species. We find that this mechanism to ensure that the enzyme is not active on translating long mRNAs is conserved from yeast to humans. Finally, we show that the products that the exosome releases after 3' to 5' degradation of the mRNA body are indeed short enough to be decapped by DcpS. Our data thus directly confirms the notion that mRNA products of the exosome are direct substrates for DcpS. In summary, we demonstrate a direct relationship between conformational changes and enzyme activity that is exploited to achieve substrate selectivity.