Biology subjects

Friedländer, M. R.

Publications and source records attributed to Friedländer, M. R..

8 recordsLinked to original sources

bioRxiv2025

The mRNA architecture of the termination site primes programmed stop codon readthrough events in Drosophila

Programmed stop codon readthrough (SCR) is a form of genetic re-coding, in which a near-cognate tRNA base-pairs with a stop codon, leading to the translation of a C-terminally extended protein. Recent studies revealed that SCR represents an evolutionarily conserved, spatio-temporally controlled mechanism of posttranscriptional gene regulation that requires cis-regulatory elements as well as trans-acting factors. In this study, we characterized cis-regulatory elements controlling programmed SCR of the Drosophila POU3-family member drifter/ventral veins lacking (dfr/vvl). Using S2 cell-based luciferase assays, we show that stop codon identity and the +4 to +9 nucleotide sequence are required but not sufficient for dfr SCR regulation. Phylogenetic prediction identified an mRNA stem-loop in the 3 UTR, proximal to the readthrough UAG codon. Mutational analysis revealed that the distance from the stop codon as well as stem-loop stability, but not the underlying sequence identity, critically impact dfr SCR. Similarly, the mRNA stem-loop promoted SCR in an in vivo Drosophila model. We applied this information to refine computational prediction of SCR-associated mRNA stem-loops and show that these elements effectively promote SCR of heterologous mRNAs. These findings increase our understanding of SCR and the underlying regulatory mechanisms. Key PointsO_LIStop codon readthrough is regulated both by mRNA sequence identity and mRNA structure elements. C_LIO_LIA 3 mRNA stem-loop and its thermodynamic stability determine stop codon readthrough efficiency. C_LIO_LImRNA stem-loops are frequently found in Drosophila genes that exhibit stop codon readthrough. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/685291v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@dbccf6org.highwire.dtl.DTLVardef@6d53f3org.highwire.dtl.DTLVardef@1a27deborg.highwire.dtl.DTLVardef@f8e5ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

SURFR: genome-free discovery of cancer-unique small RNAs from cohort sequencing data

Small RNAs play numerous roles in cancer biology and function as biomarkers and therapeutic targets. A recently discovered class of small RNAs - the orphan non-coding RNAs (oncRNAs) - are uniquely present in cancer and absent from healthy tissues. Here, we developed a reference-free computational approach that is independent from the human genome and applied it on cohort sequencing data from five cancer types to discover 1211 novel oncRNAs. Most of these new sequences do not map perfectly to the human genome, and hundreds correlate with patient outcomes. Our new oncRNAs include the first bona fide cancer-unique microRNA; oncRNAs predictive of patient survival and a small RNA that is expressed from an evolutionarily ancestral part of the genome that has been lost in most human individuals. In summary, we provide our reference-free computational approach and more than thousand new cancer-unique RNAs as resources to the community.

genomics↗

bioRxiv2024

Landscape of microRNA and target expression variation and covariation in single mouse embryonic stem cells

MicroRNAs are small RNA molecules that can repress the expression of protein coding genes post-transcriptionally. Previous studies have shown that microRNAs can also have alternative functions including target noise buffering and co-expression, but these observations have been limited to a few microRNAs. Here we systematically study microRNA alternative functions in mouse embryonic stem cells, by genetically deleting Drosha - leading to global loss of microRNAs. We apply complementary single-cell RNA-seq methods to study the variation of the targets and the microRNAs themselves, and transcriptional inhibition to measure target half-lives. We find that microRNAs form four distinct co-expression groups across single cells. In particular the mir-290 and the mir-182 clusters are abundantly, variably and inversely expressed. Intriguingly, some cells have global biases towards specific miRNAs originating from either end of the hairpin precursor, suggesting the presence of unknown regulatory cofactors. We find that miRNAs generally increase variation and covariation of their targets at the RNA level, but we also find miRNAs such as miR-182 that appear to have opposite functions. In particular, miRNAs that are themselves variable in expression, such as miR-291a, are more likely to induce covariations. In summary, we apply genetic perturbation and multi-omics to give the first global picture of microRNA dynamics at the single cell level.

genomics↗

bioRxiv2022

agoTRIBE detects miRNA-target interactions transcriptome-wide in single cells

MicroRNAs are gene regulatory molecules that play important roles in numerous biological processes including human health. The function of a given microRNA is defined by its selection of target transcripts, yet current state-of-the-art experimental methods to identify microRNA targets are laborious and require millions of cells. We have overcome these limitations by fusing the microRNA effector protein Argonaute2 to the RNA editing domain of ADAR2, allowing for the first time the detection of microRNA targets transcriptome-wide in single cells. Our agoTRIBE method reports functional microRNA targets which are additionally supported by evolutionary sequence conservation. As a proof-of-principle, we study microRNA interactions in single cells, and find substantial differential targeting across the cell cycle. Lastly, agoTRIBE additionally provides transcriptome-wide measurements of RNA abundance and will allow the deconvolution of microRNA targeting in complex samples such as tissues at the single-cell level.

genomics↗

bioRxiv2021

MapToCleave: high-throughput profiling of microRNA biogenesis in living cells

Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors, however, they have been conducted in vitro. Here we introduce MapToCleave, a new method to simultaneously profile processing of thousands of distinct RNA structures in living cells. Our new in cell method captures essentially all the biogenesis features that have been discovered through near two decades of in vitro studies - providing support for both approaches. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from twenty animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from ten animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryo-EM studies. In summary, we apply a new screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.

molecular biology↗

bioRxiv2021

Environmental enrichment induces epigenomic and genome organization changes relevant for cognitive function

In early development, the environment triggers mnemonic epigenomic programs resulting in memory and learning experiences to confer cognitive phenotypes into adulthood. To uncover how environmental stimulation impacts the epigenome and genome organization, we used the paradigm of environmental enrichment (EE) in young mice constantly receiving novel stimulation. We profiled epigenome and chromatin architecture in whole cortex and sorted neurons by deep-sequencing techniques. Specifically, we studied chromatin accessibility, gene and protein regulation, and 3D genome conformation, combined with predicted enhancer and chromatin interactions. We identified increased chromatin accessibility, transcription factor binding including CTCF-mediated insulation, differential occupancy of H3K36me3 and H3K79me2, and changes in transcriptional programs required for neuronal development. EE stimuli led to local genome re-organization by inducing increased contacts between chromosomes 7 and 17 (inter-chromosomal). Our findings support the notion that EE-induced learning and memory processes are directly associated with the epigenome and genome organization. Highlights- Environmental enrichment (EE) alters chromatin conformation, CTCF binding, and spatially 3D genome changes, thereby regulating cognitive function during the first steps of life after birth. - Transcription-associated gene body marks H3K79me2 and H3K36me3 are differently influenced by EE in cortical brain cells and binding is exacerbated upon stimulation in an age-dependent manner. - EE-induced changes of 3D genome organization increase inter-chromosomal interactions of genes associated with synaptic transmission and AMPA receptor genes on chromosomes 7 and 17.

neuroscience↗

bioRxiv2019

The SWI/SNF subunits BRG1 affects alternative splicing by changing RNA binding factor interactions with RNA

BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes. The function of the SWI/SNF complexes in transcriptional initiation has been well studied, while a function in alternative splicing has only been studied for a few cases for BRM-containing SWI/SNF complexes. Here, we have expressed BRG1 in C33A cells, a BRG1 and BRM-deficient cell line, and we have analysed the effects on the transcriptome by RNA sequencing. We have shown that BRG1 expression affects the splicing of a subset of genes. For some, BRG1 expression favours exon inclusion and for others, exon skipping. Some of the changes in alternative splicing induced by BRG1 expression do not require the ATPase activity of BRG1. Among the exons regulated through an ATPase-independent mechanism, the included exons had signatures of high GC-content and lacked a positioned nucleosome at the exon. By investigating three genes in which the expression of either wild-type BRG1 or a BRG1-ATPase-deficient variant favoured exon inclusion, we showed that expression of the ATPases promotes the local recruitment of RNA binding factors to chromatin and RNA in a differential manner. The hnRNPL, hnRNPU and SAM68 proteins associated to chromatin in C33A cells expressing BRG1 or BRM, but their association with RNA varied. We propose that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and altering their binding to the nascent pre-mRNA, which changes RNP structure. Author summarySplicing, in particular alternative splicing, is a combinatorial process which involves splicing factor complexes and many RNA binding splicing regulatory proteins in different constellations. Most splicing events occur during transcription, which also makes the DNA sequence, the chromatin state and the transcription rate at the exons important components that influence the splicing outcome. We show here that the ATP-dependent chromatin remodelling complex SWI/SNF influences the interactions of splicing regulatory factors with RNA during transcription on certain exons that have a high GC-content. The splicing on this type of exon rely on the ATPase BRG1 and favour inclusion of alternative exons in an ATP-independent manner. SWI/SNF complexes are known to alter the chromatin structure at promoters in transcription initiation, and have been previously shown to alter the transcription rate or nucleosome position in splicing. Our results suggests a further mechanism for chromatin remodelling proteins in splicing: to change the interaction patterns of RNA binding splicing regulatory factors at alternative exons to alter the splicing outcome.

molecular biology↗

bioRxiv2019

Nuclear gene proximity and protein interactions shape transcript covariances in mammalian single cells

Single-cell RNA sequencing studies into gene co-expression patterns could yield important new regulatory and functional insights, but have so far been limited by the confounding effects of cell differentiation and the cell cycle. We apply a tailored experimental design that eliminates these confounders, and report >80,000 intrinsically covarying gene pairs in mouse embryonic stem cells. These covariances form a network with biological properties, outlining known and novel gene interactions. We provide the first evidence that miRNAs naturally induce transcriptome-wide covariances, and compare the relative importance of nuclear organization, transcriptional and post-transcriptional regulation in defining covariances. We find that nuclear organization has the greatest impact, and that genes encoding for physically interacting proteins specifically tend to covary, suggesting importance for protein complex stoichiometry. Our results lend support to the concept of post-transcriptional RNA operons, but we further present evidence that nuclear proximity of genes on the same or even distinct chromosomes also provides substantial functional regulation in mammalian single cells.

systems biology↗