View source for Ribonucleoprotein Networks Analyzed by Mutational Profiling

{{Short description|Protein-RNA binding probing method}}
{{Orphan|date=August 2022}}

[[Ribonucleoprotein|'''R'''ibo'''n'''ucleo'''p'''rotein]] Networks Analyzed by '''M'''ut'''a'''tional '''P'''rofiling '''(RNP-MaP)''' is a strategy for probing [[RNA]]-[[protein]] networks and protein binding sites at a [[nucleotide]] resolution. Information about RNP assembly and function can facilitate a better understanding of biological mechanisms. RNP-MaP uses [[N-Hydroxysuccinimide|NHS]]-[[diazirine]] (SDA), a hetero-bifunctional crosslinker, to freeze RNA-bound proteins in place. Once the RNA-protein [[Cross-link|crosslinks]] are formed, MaP [[reverse transcription]]<ref name=":0">{{Cite journal |last1=Smola |first1=Matthew J. |last2=Rice |first2=Greggory M. |last3=Busan |first3=Steven |last4=Siegfried |first4=Nathan A. |last5=Weeks |first5=Kevin M. |date=2015-10-01 |title=Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis |journal=Nature Protocols |language=en |volume=10 |issue=11 |pages=1643–1669 |doi=10.1038/nprot.2015.103 |pmid=26426499 |pmc=4900152 |issn=1750-2799}}</ref> is then conducted to reversely transcribe the protein-bound RNAs as well as introduce mutations at the site of RNA-protein crosslinks. Sequencing results of the [[Complementary DNA|cDNAs]] reveal information about both protein-RNA interaction networks and protein binding sites.<ref name=":1">{{Cite journal |last1=Weidmann |first1=Chase A. |last2=Mustoe |first2=Anthony M. |last3=Jariwala |first3=Parth B. |last4=Calabrese |first4=J. Mauro |last5=Weeks |first5=Kevin M. |date=2020-10-19 |title=Analysis of RNA–protein networks with RNP-MaP defines functional hubs on RNA |journal=Nature Biotechnology |language=en |volume=39 |issue=3 |pages=347–356 |doi=10.1038/s41587-020-0709-7 |pmid=33077962 |pmc=7956044 |issn=1546-1696}}</ref>
[[File:RNP-MaP workflow.png|thumb|none|900x1000px|RNP-MaP workflow overview]]

== Strategy ==

=== Components ===
RNA-MaP involves three major components:<ref name=":1" />

* Ribonucleoproteins (RNPs): complexes made up of RNAs and [[RNA-binding protein]]s (RBPs)<ref name=":3">{{Cite journal |last1=Stefl |first1=Richard |last2=Skrisovska |first2=Lenka |last3=Allain |first3=Frédéric H.-T. |date=January 2005 |title=RNA sequence- and shape-dependent recognition by proteins in the ribonucleoprotein particle |journal=EMBO Reports |language=en |volume=6 |issue=1 |pages=33–38 |doi=10.1038/sj.embor.7400325 |pmid=15643449|pmc=1299235 }}</ref>
* NHS-diazirine (SDA): a cell permeable crosslinking reagent. SDA contains two reactive groups - a diazirine and a succinimidyl [[ester]]. The reaction between succinimidyl esters and [[amine]] groups (e.g. [[lysine]] side chains) results in [[peptide bond]]s (or [[amide]] bonds). When exposed to [[Ultraviolet|UV light]] with a wavelength of 365&nbsp;nm, an intermediate broadly reactive toward nucleotide [[ribose]]s and bases is formed. As a result, proteins are crosslinked with RNA by the SDA linker.<ref name=":1" />
* Mutational profiling (MaP): a method using reverse transcriptase with relaxed fidelity to incorporate modified residues at protein-RNA binding sites.<ref name=":0" /><ref name=":3" />

=== Workflow ===
Long-wavelength UV and SDA reagents are first supplied to living cells to crosslink protein residues with RNA by forming amide bonds between amine groups of lysine (or [[arginine]]) residues and succinimidyl esters. Next, cells containing crosslinked RNPs are [[Lysis|lysed]] and the RNA-bound proteins are digested into peptide adducts. MaP reverse transcription is then performed to label the protein-RNA binding sites through peptide adduct-induced [[mutation]]s. [[Sequencing]] of the mutation-containing cDNA product will reveal the mutation sites (or RNP-MaP sites) and the correlations between the RNP-MaP sites are computationally determined using 3-nucleotide windows.<ref name=":1" /><ref name=":2">{{Cite journal |last1=Mustoe |first1=Anthony M. |last2=Lama |first2=Nicole N. |last3=Irving |first3=Patrick S. |last4=Olson |first4=Samuel W. |last5=Weeks |first5=Kevin M. |date=2019-11-19 |title=RNA base-pairing complexity in living cells visualized by correlated chemical probing |journal=Proceedings of the National Academy of Sciences |volume=116 |issue=49 |pages=24574–24582 |doi=10.1073/pnas.1905491116 |issn=0027-8424 |pmc=6900531 |pmid=31744869|bibcode=2019PNAS..11624574M |doi-access=free }}</ref>

== Analysis ==

=== RNP-MaP site identification ===
RNP-MaP sites are defined as protein bound nucleotides. SDA and UV treated and UV only treated sample sequence reads are aligned and mutations are counted using ShapeMapper2 software.<ref>{{Cite journal |last1=Busan |first1=Steven |last2=Weeks |first2=Kevin M. |date=2018-02-01 |title=Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2 |url=http://rnajournal.cshlp.org/content/24/2/143 |journal=RNA |language=en |volume=24 |issue=2 |pages=143–148 |doi=10.1261/rna.061945.117 |issn=1355-8382 |pmc=5769742 |pmid=29114018}}</ref> The SDA or RNP-MaP reactivity for a nucleotide is the ratio of the crosslinked (SDA and UV treated) mutational frequency to the un-crosslinked (UV only) mutation frequency. Using differential mutational signatures, RNP-MaP sites are identified based on universal [[Normalization (statistics)|normalization]] factors and thresholds on each RNA nucleotide (U, A, C, and G) derived from analysis of ribonucleoproteins of known structure.<ref name=":1" />

A nucleotide is identified as a RNP-MaP site if it passes three filters:

# The number of mutation events in the SDA + UV-treated sample is at least 50 greater than the UV-treated sample.
# Site reactivities must exceed the nucleotide-dependent thresholds (<math>T_x</math>) that are empirically defined as:
#* <math>T_x = \frac{BG_{X>10} - MED_{all X}}{SD_{all X}}</math>, where
#** <math>X</math> is the nucleotide U, A, C, or G
#** <math>BG_X</math> is the background threshold defined by the 90% reactivity values of nucleotides in a >10Å group
#** <math>MED_{all X}</math> is the median of all reactivities for all nucleotides
#** <math>SD_{all X}</math> is the standard deviation of reactivities for all nucleotides
# The calculated Z-factor is greater than zero
#* The calculated Z-factor is defined as
#** <math>Z-factor = 1-\frac{2.575(\sigma_{SDA+UV}+\sigma_{UV})}{|\text{mutation rate}_{SDA+UV}-\text{mutation rate}_{UV}|}</math>, where
#*** <math>\text{mutation rate}_{SDA+UV}</math> is the mutation rate of a nucleotide treated with SDA and UV and <math>\text{mutation rate}_{UV}</math> is the mutation rate of a nucleotide treated with UV only
#*** <math>\sigma_{nt} = \frac{\sqrt{\text{mutation rate}_{nt}}}{\sqrt{\text{reads}_{nt}}}</math>, where <math>nt</math> is treatment (SDA + UV or UV only)

=== Protein-RNA interaction network identification ===
Protein-RNA interactions networks are identified using RNP-MaP [[correlation]]s since multiple crosslink sites can be detected for a single RNA molecule. RNP-MaP correlations provide a complementary measure of protein binding to RNA independent of RNP-MaP sites.<ref name=":1" /> They are identified using a G-test framework known as RingMapper.<ref name=":2" />

RNP-MaP correlations require a single RNA molecule to form at least two crosslinks and arise from any of three scenarios:

# A single protein binds to two locations of one RNA
# Two proteins that interact and bind to two locations on one RNA
# Two proteins are deposited on two locations on one RNA by a coordinated assembly process

Using RNP-MaP correlations, a network of protein-RNA interaction sites is found and can then be used for functional analysis.

== Related methods ==

=== Cross-linking immunoprecipitation (CLIP) ===
[[Cross-linking immunoprecipitation|CLIP]] analyzes protein interactions with RNA by combining UV cross-linking and [[immunoprecipitation]]. CLIP-based techniques are able to map RNA binding protein binding sites of interest on a genome-wide scale.<ref>{{Cite journal |last1=Hafner |first1=Markus |last2=Katsantoni |first2=Maria |last3=Köster |first3=Tino |last4=Marks |first4=James |last5=Mukherjee |first5=Joyita |last6=Staiger |first6=Dorothee |last7=Ule |first7=Jernej |last8=Zavolan |first8=Mihaela |date=2021-03-04 |title=CLIP and complementary methods |journal=Nature Reviews Methods Primers |language=en |volume=1 |issue=1 |pages=1–23 |doi=10.1038/s43586-021-00018-1 |s2cid=233834798 |issn=2662-8449|doi-access=free }}</ref>

There are many CLIP-based methods including:

* [[HITS-CLIP]] ([[High throughput sequencing|High-throughput sequencing]] of RNA isolated by crosslinking immunoprecipitation or CLIP-seq)<ref>{{Cite journal |last=Darnell |first=Robert B. |date=2010-08-02 |title=HITS-CLIP: panoramic views of protein–RNA regulation in living cells |journal=Wiley Interdisciplinary Reviews: RNA |language=en |volume=1 |issue=2 |pages=266–286 |doi=10.1002/wrna.31 |pmid=21935890 |pmc=3222227 |issn=1757-7004}}</ref>
* [[PAR-CLIP]] (Photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation)<ref>{{Cite journal |last1=Hafner |first1=Markus |last2=Landthaler |first2=Markus |last3=Burger |first3=Lukas |last4=Khorshid |first4=Mohsen |last5=Hausser |first5=Jean |last6=Berninger |first6=Philipp |last7=Rothballer |first7=Andrea |last8=Ascano |first8=Manuel |last9=Jungkamp |first9=Anna-Carina |last10=Munschauer |first10=Mathias |last11=Ulrich |first11=Alexander |date=2010-07-02 |title=PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins |url=http://www.jove.com/index/Details.stp?ID=2034 |journal=Journal of Visualized Experiments |language=en |issue=41 |pages=2034 |doi=10.3791/2034 |issn=1940-087X |pmc=3156069 |pmid=20644507}}</ref>
* [[iCLIP]] (Individual nucleotide-resolution cross-linking and immunoprecipitation)<ref>{{Cite journal |last1=König |first1=Julian |last2=Zarnack |first2=Kathi |last3=Rot |first3=Gregor |last4=Curk |first4=Tomaž |last5=Kayikci |first5=Melis |last6=Zupan |first6=Blaž |last7=Turner |first7=Daniel J. |last8=Luscombe |first8=Nicholas M. |last9=Ule |first9=Jernej |date=2010-07-04 |title=iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution |journal=Nature Structural & Molecular Biology |language=en |volume=17 |issue=7 |pages=909–915 |doi=10.1038/nsmb.1838 |pmid=20601959 |pmc=3000544 |issn=1545-9985}}</ref>
* eCLIP (Enhanced cross-linking and immunoprecipitation followed by high-throughput sequencing)<ref>{{Cite journal |last1=Van Nostrand |first1=Eric L |last2=Pratt |first2=Gabriel A |last3=Shishkin |first3=Alexander A |last4=Gelboin-Burkhart |first4=Chelsea |last5=Fang |first5=Mark Y |last6=Sundararaman |first6=Balaji |last7=Blue |first7=Steven M |last8=Nguyen |first8=Thai B |last9=Surka |first9=Christine |last10=Elkins |first10=Keri |last11=Stanton |first11=Rebecca |date=2016-03-28 |title=Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP) |journal=Nature Methods |language=en |volume=13 |issue=6 |pages=508–514 |doi=10.1038/nmeth.3810 |issn=1548-7091 |pmc=4887338 |pmid=27018577}}</ref>
* sCLIP (Simple cross-linking and immunoprecipitation)<ref>{{Cite journal |last1=Kargapolova |first1=Yulia |last2=Levin |first2=Michal |last3=Lackner |first3=Karl |last4=Danckwardt |first4=Sven |date=2017-06-02 |title=sCLIP—an integrated platform to study RNA–protein interactomes in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs |url=https://academic.oup.com/nar/article/45/10/6074/3057353 |journal=Nucleic Acids Research |language=en |volume=45 |issue=10 |pages=6074–6086 |doi=10.1093/nar/gkx152 |issn=0305-1048 |pmc=5449641 |pmid=28334977}}</ref>

=== Mass spectrometry ===
Quantitative [[mass spectrometry]] (MS) (or [[quantitative proteomics]]) can be used to discover RNA-binding proteins (RBPs) bound to RNA. Labeling MS methods involve the differential use of stable isotope labels or chemical tagging of proteins in samples and controls. This is used to obtain enrichment scores and true binding partners through the ratio of labeled peptides.<ref>{{Cite journal |last1=Bantscheff |first1=Marcus |last2=Schirle |first2=Markus |last3=Sweetman |first3=Gavain |last4=Rick |first4=Jens |last5=Kuster |first5=Bernhard |date=2007-10-01 |title=Quantitative mass spectrometry in proteomics: a critical review |journal=Analytical and Bioanalytical Chemistry |language=en |volume=389 |issue=4 |pages=1017–1031 |doi=10.1007/s00216-007-1486-6 |pmid=17668192 |s2cid=14236144 |issn=1618-2650|doi-access=free }}</ref> Label-free MS methods are able to identify proteins in samples and controls. In order to distinguish true binding partners for nonspecific proteins, analytical tools used alongside spectral count data from non-quantitative MS are used to score the probability of a true RBP-RNA interaction<ref>{{Cite journal |last1=Mellacheruvu |first1=Dattatreya |last2=Wright |first2=Zachary |last3=Couzens |first3=Amber L. |last4=Lambert |first4=Jean-Philippe |last5=St-Denis |first5=Nicole A. |last6=Li |first6=Tuo |last7=Miteva |first7=Yana V. |last8=Hauri |first8=Simon |last9=Sardiu |first9=Mihaela E. |last10=Low |first10=Teck Yew |last11=Halim |first11=Vincentius A. |date=2013-07-07 |title=The CRAPome: a contaminant repository for affinity purification–mass spectrometry data |journal=Nature Methods |language=en |volume=10 |issue=8 |pages=730–736 |doi=10.1038/nmeth.2557 |pmid=23921808 |pmc=3773500 |issn=1548-7105}}</ref>

== Advantages and Limitations ==

=== Advantages ===
RNP-MaP can help reveal functionally important RNA-protein binding networks through binding site density and interconnectivity independent of previous knowledge of interacting proteins. Because of the unbiased nature of the analysis, RNP-MaP is able to detect conserved RNA-protein interactions between species.<ref name=":1" />

RNP-MaP is also able to facilitate the characterization of functionally critical elements in large non-coding RNAs or even viral RNAs.<ref name=":1" />

=== Limitations ===
As a standalone technique, RNP-MaP cannot be used to determine protein-RNA binding mechanisms or protein identities. In order to do so, RNP-MaP must be used in conjunction with other techniques such as CLIP and mass spectrometry.<ref name=":1" />

RNP-MaP requires extremely high [[Read depth|read-depths]] for analysis. To identify RNP-MaP sites, 1000x [[sequencing coverage]] is required, while RNP-MaP correlation sites require 10,000x sequencing coverage.<ref name=":1" />

There are severe limitations on the ability to characterize RNP-MaP correlations between distant (>500 nucleotides) RNP-MaP sites. This is due to limitations of MaP reverse transcription processivity (500-600 nucleotides) and sequencing instrument clustering (<1,000 nucleotides).<ref name=":1" />

== References ==

{{reflist}}



[[Category:Genetics techniques]]
[[Category:RNA]]