RNA-GPS predicts high-resolution RNA subcellular localization and highlights the role of splicing

Wu KE, Parker KR, Fazal FM, Chang HY, Zhou J.

RNA, 2020

doi:10.1261/rna.074161.119

Subcellular localization is essential to RNA biogenesis, processing, and function across the gene expression life cycle. However, the specific nucleotide sequence motifs that direct RNA localization are incompletely understood. Fortunately, new sequencing technologies have provided transcriptome-wide atlases of RNA localization, creating an opportunity to leverage computational modeling. Here we present RNA-GPS, a new machine learning model that uses nucleotide-level features to predict RNA localization across eight different subcellular locations-the first to provide such a wide range of predictions. RNA-GPS's design enables high-throughput sequence ablation and feature importance analyses to probe the sequence motifs that drive localization prediction. We find localization informative motifs to be concentrated on 3'-UTRs and scattered along the coding sequence, and motifs related to splicing to be important drivers of predicted localization, even for cytotopic distinctions for membraneless bodies within the nucleus or for organelles within the cytoplasm. Overall, our results suggest transcript splicing is one of many elements influencing RNA subcellular localization.

RNA-GPS predicts SARS-CoV-2 RNA residency to host mitochondria and nucleolus

Wu KE, Fazal FM, Parker KR, Zou J, Chang HY.

Cell Systems, 2020

doi:10.1016/j.cels.2020.06.008

SARS-CoV-2 genomic and subgenomic RNA (sgRNA) transcripts hijack the host cell's machinery. Subcellular localization of its viral RNA could, thus, play important roles in viral replication and host antiviral immune response. We perform computational modeling of SARS-CoV-2 viral RNA subcellular residency across eight subcellular neighborhoods. We compare hundreds of SARS-CoV-2 genomes with the human transcriptome and other coronaviruses. We predict the SARS-CoV-2 RNA genome and sgRNAs to be enriched toward the host mitochondrial matrix and nucleolus, and that the 5' and 3' viral untranslated regions contain the strongest, most distinct localization signals. We interpret the mitochondrial residency signal as an indicator of intracellular RNA trafficking with respect to double-membrane vesicles, a critical stage in the coronavirus life cycle. Our computational analysis serves as a hypothesis generation tool to suggest models for SARS-CoV-2 biology and inform experimental efforts to combat the virus. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.

Subcellular spatial transcriptomes: emerging frontier for understanding gene regulation

Fazal FM, Chang HY.

Cold Spring Harbor Symposium on Quantitative Biology, 2020

doi:10.1101/sqb.2019.84.040352

RNAs are trafficked and localized with exquisite precision inside the cell. Studies of candidate messenger RNAs have shown the vital importance of RNA subcellular location in development and cellular function. New sequencing- and imaging-based methods are providing complementary insights into subcellular localization of RNAs transcriptome-wide. APEX-seq and ribosome profiling as well as proximity-labeling approaches have revealed thousands of transcript isoforms are localized to distinct cytotopic locations, including locations that defy biochemical fractionation and hence were missed by prior studies. Sequences in the 3' and 5' untranslated regions (UTRs) serve as "zip codes" to direct transcripts to particular locales, and it is clear that intronic and retrotransposable sequences within transcripts have been co-opted by cells to control localization. Molecular motors, nuclear-to-cytosol RNA export, liquid-liquid phase separation, RNA modifications, and RNA structure dynamically shape the subcellular transcriptome. Location-based RNA regulation continues to pose new mysteries for the field, yet promises to reveal insights into fundamental cell biology and disease mechanisms.

RNA structure maps across mammalian cellular compartments

Sun L*, Fazal FM*, Li P*, Broughton JP, Lee B, Tang L, Huang W, Kool ET, Chang HY, Zhang QC.

Nature Structural and Molecular Biology (NSMB), 2019

doi:10.1038/s41594-019-0200-7

RNA structure is intimately connected to each step of gene expression. Recent advances have enabled transcriptome-wide maps of RNA secondary structure, called 'RNA structuromes'. However, previous whole-cell analyses lacked the resolution to unravel the landscape and also the regulatory mechanisms of RNA structural changes across subcellular compartments. Here we reveal the RNA structuromes in three compartments, chromatin, nucleoplasm and cytoplasm, in human and mouse cells. The cytotopic structuromes substantially expand RNA structural information and enable detailed investigation of the central role of RNA structure in linking transcription, translation and RNA decay. We develop a resource with which to visualize the interplay of RNA-protein interactions, RNA modifications and RNA structure and predict both direct and indirect reader proteins of RNA modifications. We also validate a novel role for the RNA-binding protein LIN28A as an N6-methyladenosine modification 'anti-reader'. Our results highlight the dynamic nature of RNA structures and its functional importance in gene regulation.

Covered by NSMB News and Views and Tsinghua Coverage

Other Links: Data Tracks Human and Mouse, bioRxiv

Real-time observation of polymerase-promoter contact remodeling during transcription initiation

Meng CA*, Fazal FM*, Block SM

Nature Communications, 2017

doi:10.1038/s41467-017-01041-1

Critical contacts made between the RNA polymerase (RNAP) holoenzyme and promoter DNA modulate not only the strength of promoter binding, but also the frequency and timing of promoter escape during transcription. Here, we describe a single-molecule optical-trapping assay to study transcription initiation in real time, and use it to map contacts formed between σ70 RNAP holoenzyme from E. coli and the T7A1 promoter, as well as to observe the remodeling of those contacts during the transition to the elongation phase. The strong binding contacts identified in certain well-known promoter regions, such as the -35 and -10 elements, do not necessarily coincide with the most highly conserved portions of these sequences. Strong contacts formed within the spacer region (-10 to -35) and with the -10 element are essential for initiation and promoter escape, respectively, and the holoenzyme releases contacts with promoter elements in a non-sequential fashion during escape.

LncRNA structure: message to the heart

Fazal FM, Chang HY.

Molecular Cell, 2016

doi:10.1016/j.molcel.2016.09.030

In this issue, Xue et al. (2016) describe the secondary structure of the heart-specific long non-coding RNA Braveheart, leading to the discovery of a short, asymmetric G-rich loop that controls cardiac lineage commitment by interacting with the transcription factor CNBP.

Direct observation of processive exoribonuclease motion using optical tweezers

Fazal FM*, Koslover DJ*, Luisi BF, Block SM.

Proceedings of the National Academy of Sciences (PNAS), 2015

doi:10.1073/pnas.1514028112

Bacterial RNases catalyze the turnover of RNA and are essential for gene expression and quality surveillance of transcripts. In Escherichia coli, the exoribonucleases RNase R and polynucleotide phosphorylase (PNPase) play critical roles in degrading RNA. Here, we developed an optical-trapping assay to monitor the translocation of individual enzymes along RNA-based substrates. Single-molecule records of motion reveal RNase R to be highly processive: one molecule can unwind over 500 bp of a structured substrate. However, enzyme progress is interrupted by pausing and stalling events that can slow degradation in a sequence-dependent fashion. We found that the distance traveled by PNPase through structured RNA is dependent on the A+U content of the substrate and that removal of its KH and S1 RNA-binding domains can reduce enzyme processivity without affecting the velocity. By a periodogram analysis of single-molecule records, we establish that PNPase takes discrete steps of six or seven nucleotides. These findings, in combination with previous structural and biochemical data, support an asymmetric inchworm mechanism for PNPase motion. The assay developed here for RNase R and PNPase is well suited to studies of other exonucleases and helicases.

Real-time observation of the initiation of RNA polymerase II transcription

Fazal FM*, Meng CA*, Murakami K*, Kornberg RD, Block SM.

Nature, 2015

doi:10.1038/nature14882

Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.

Covered by Stanford News, Biotechniques, Phys.org, and Yeast Genome Database

Binding and translocation of termination factor Rho studied at the single-molecule level

Koslover DJ*, Fazal FM*, Mooney RA, Landick R, Block SM.

Journal of Molecular Biology (JMB), 2012

doi:10.1016/j.jmb.2012.07.027

Rho termination factor is an essential hexameric helicase responsible for terminating 20-50% of all mRNA synthesis in Escherichia coli. We used single-molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho utilization site of the λtR1 terminator. Our results are consistent with Rho complexes adopting two states: one that binds 57 ± 2nt of RNA across all six of the Rho primary binding sites, and another that binds 85 ± 2nt at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5'→3' toward RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the Rho utilization site and RNAP. These findings lead to a general model for Rho binding and translocation and establish a novel experimental approach that should facilitate additional single-molecule studies of RNA-binding proteins.

Other Links: JMB Commentary, Featured Article and Cover

Optical tweezers study life under tension

Fazal FM, Block SM.

Nature Photonics, 2011

doi:10.1038/nphoton.2011.100

Optical tweezers have become one of the primary weapons in the arsenal of biophysicists, and have revolutionized the new field of single-molecule biophysics. Today's techniques allow high-resolution experiments on biological macromolecules that were mere pipe dreams only a decade ago.

Other Links: Nobel Prize in Physics 2018 Nature Collection

Spectral energy distributions of high-mass protostellar objects – evidence for high accretion rates

Fazal FM, Sridharan TK, Qui K, Whitney B, Zhang Q, Robitaille T.

Astrophysical Journal Letters (ApJL), 2008

doi:10.1086/593975

The spectral energy distributions (SEDs), spanning the mid-infrared to millimeter wavelengths, of a sample of 13 high-mass protostellar objects (HMPOs) were studied using a large archive of 2D axisymmetric radiative transfer models. Measurements from the Spitzer GLIMPSE and MIPSGAL surveys and the MSX survey were used in addition to our own surveys at millimeter and submillimeter wavelengths to construct the SEDs, which were then fit to the archive of models. These models assumed that stars of all masses form via accretion and allowed us to make estimates for the masses, luminosities, and envelope accretion rates for the HMPOs. The models fit the observed SEDs well. The implied envelope accretion rates are high, ≈10−2.5M☉ yr−1, consistent with the accretion-based scenario of massive star formation. With the fitted accretion rates and with mass estimates of up to ~20 M☉ for these objects, it appears plausible that stars with stellar masses M* > 20 M☉ can form via accretion.

Circular differential double diffraction in chiral media

Ghosh A, Fazal FM, Fischer P.

Optics Letters, 2007

doi:10.1364/OL.32.001836

In an optically active liquid the diffraction angle depends on the circular polarization state of the incident light beam. We report the observation of circular differential diffraction in an isotropic chiral medium, and we demonstrate that double diffraction is an alternate means to determine the handedness (enantiomeric excess) of a solution.

Glucose-specific poly(allylamine) hydrogels – a reassessment

Fazal FM, Hansen DE.

Bioorganic and Medicinal Chemistry Letters (BMCL), 2007

doi:10.1016/j.bmcl.2006.09.054

Polymer hydrogels synthesized by crosslinking poly(allylamine hydrochloride) with (±)-epichlorohydrin in the presence of d-glucose-6-phosphate monobarium salt do not show imprinting on the molecular level. A series of hydrogels was prepared using the following five templates: d-glucose-6-phosphate monobarium salt, d-glucose, l-glucose, barium hydrogen phosphate (BaHPO4), and d-gluconamide; a hydrogel was also prepared in the absence of a template. For all six hydrogels, batch binding studies were conducted with d-glucose, l-glucose, d-fructose, and d-gluconamide. The extent of analyte sugar binding was determined using 1H NMR. Each hydrogel shows approximately the same relative binding affinity for the different sugar derivatives, and none displays selectivity for either glucose enantiomer. The results of the binding studies correlate with the octanol–water partition coefficients of the sugars, indicative that differential solubilities in the bulk polymer account for the binding affinities observed. Thus, in contrast to templated hydrogels prepared using methacrylate- or acrylamide-based reagents, true imprinting does not occur in this novel, crosslinked-poly(allylamine hydrochloride) system.