Biology subjects

Saleem, I.

Publications and source records attributed to Saleem, I..

4 recordsLinked to original sources

bioRxiv2025

ECD functions as a novel RNA-binding protein to regulate mRNA splicing

Human ecdysoneless protein (ECD) plays an essential role in regulating cell cycle progression and cell survival. ECD has previously been implicated in RNA splicing through its association with spliceosomal proteins. Here, using EMSA, fluorescence polarization assays, and mutational analysis, we demonstrate that ECD directly binds to RNA. Enhanced CLIP-seq analysis identified a broad repertoire of mRNAs bound to ECD in cells. RNA-seq analyses revealed that ECD depletion leads to widespread splicing aberrations and altered gene expression. ECD binding to RNAs was enriched near splice sites, and a substantial fraction of ECD-bound transcripts exhibited splicing defects upon ECD depletion. ECD associates with and stabilizes the U5 snRNP complex specific proteins. While depletion of ECD reduced the levels of key U5-specifc proteins, these proteins exhibited an increased association with the R2TP complex in knockout cells. Notably, we found ECD to directly bind to U5 snRNA, and an RNA binding defective mutant of ECD ({Delta}135-148) failed to rescue the reduced levels of U5-specific proteins or the proliferation defect induced by ECD depletion. Collectively, these findings demonstrate that ECD binds to RNAs, including the U5 snRNA, and that RNA-binding is required for ECD to stabilize the U5 snRNP and for cellular functions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/634785v4_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@f5aa4forg.highwire.dtl.DTLVardef@80606borg.highwire.dtl.DTLVardef@3a4c8eorg.highwire.dtl.DTLVardef@1780b0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Simultaneous mapping of RNA secondary, tertiary, and quaternary structure in living cells by multi-site DMS probing

Identifying tertiary structures and protein binding sites on RNA molecules remains a key challenge in RNA biology. We report a new chemical probing strategy termed multi-site DMS-MaP (msDMS-MaP) that exploits base tautomerization during mutational profiling reverse-transcription to measure typically invisible DMS-induced modifications at the N7 position of G. These N7-G modifications are now resolved concurrently with conventional N1 and N3 modifications with only minor modifications to established protocols, providing a multi-dimensional, single-molecule readout of RNA structure. We show that N7-G reactivity specifically reports on RNA tertiary and quaternary structure, enabling identification of diverse, functionally significant motifs such as cooperatively folding tertiary domains and protein binding sites in living cells. We apply msDMS-MaP to obtain a map of the quaternary structural ensemble of the 7SK non-coding snRNP, revealing unique protein binding sites across three 7SK structural isoforms. msDMS-MaP represents a broadly applicable strategy for enhanced RNA functional motif discovery and characterization.

biochemistry↗

bioRxiv2023

Mutation signature filtering enables high-fidelity RNA structure probing at all four nucleobases with DMS

Chemical probing experiments have transformed RNA structure analysis, enabling high-throughput measurement of base-pairing in living cells. Dimethyl sulfate (DMS) is one of the most widely used structure probing reagents and has played a prominent role in enabling next-generation single-molecule probing analyses. However, DMS has traditionally only been able to probe adenine and cytosine nucleobases. We previously showed that, using appropriate conditions, DMS can also be used to interrogate base-pairing of uracil and guanines in vitro at reduced accuracy. However, DMS remained unable to informatively probe guanines in cells. Here, we develop an improved DMS mutational profiling (MaP) strategy that leverages the unique mutational signature of N1-methylguanine DMS modifications to enable robust, high-fidelity structure probing at all four nucleotides, including in cells. Using information theory, we show that four-base DMS reactivities convey greater structural information than comparable two-base DMS and SHAPE probing strategies. Four-base DMS experiments further enable improved direct base-pair detection by single-molecule PAIR analysis, and ultimately support RNA structure modeling at superior accuracy. Four-base DMS probing experiments are easily performed and will broadly facilitate improved RNA structural analysis in living cells.

biochemistry↗

bioRxiv2021

Large-scale allosteric switch in the 7SK RNA regulates transcription in response to growth and stress

7SK is a highly conserved non-coding RNA that regulates eukaryotic transcription by sequestering positive transcription elongation factor b (P-TEFb). 7SK regulatory function likely entails changes in RNA structure, but characterizing dynamic RNA-protein complexes in cells has remained an unsolved challenge. We describe a new chemical probing strategy (DANCE-MaP) that uses maximum likelihood deconvolution and probabilistic read assignment to define simultaneously (i) per-nucleotide reactivity profiles, (ii) direct base pairing interactions, and (iii) tertiary and higher-order interactions for each conformation of multi-state RNA structural ensembles, all from a single experiment. We show that human 7SK RNA, despite significant heterogeneity, intrinsically codes for a large-scale structural switch that couples dissolution of the P-TEFb binding site to structural remodeling at distal release factor binding sites. The 7SK structural equilibrium is regulated by cell type, shifts dynamically in response to cell growth and stress, and can be exogenously targeted to modulate transcription in cells. Our data support that the 7SK structural ensemble functions as an integrator of diverse cellular signals to control transcription elongation in environment and cell specific ways, and establishes DANCE-MaP as a powerful strategy for comprehensively defining RNA structure and dynamics in cells.

biochemistry↗