Abstract
Low-complexity (LC) sequences, typically believed to be incapable of assuming structural order, are abundant constituents of the proteomes of all eukaryotic organisms. These sequences have emerged as critical components for formation of meso-scaled, sub-cellular organelles not invested by surrounding membranes, exemplified by RNA granules. We have observed that LC domains of many RNA binding proteins known to be constituents of RNA granules readily form labile cross-β polymers under physiological conditions. Several lines of experimentation have shown that formation of labile, cross-β polymers assembled from LC domain monomers is important for formation of RNA granules. Among the various experiments we have carried out, hydrogel binding assays have evolved as a versatile technique allowing a reliable means of assessing polymer formation and the binding of heterotypic cellular components integral to the formation of RNA granules. This article presents methods allowing for the production of hydrogel droplets composed of LC domain polymers. We further describe methods allowing straightforward assessment for binding of test LC domains to hydrogel droplets by fluorescence microscopy.
| Original language | English (US) |
|---|---|
| Journal | Methods |
| DOIs | |
| State | Accepted/In press - Mar 16 2017 |
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ASJC Scopus subject areas
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
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Cross-β polymerization and hydrogel formation by low-complexity sequence proteins. / Kato, Masato; Lin, Yi; McKnight, Steven L.
In: Methods, 16.03.2017.Research output: Contribution to journal › Article
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TY - JOUR
T1 - Cross-β polymerization and hydrogel formation by low-complexity sequence proteins
AU - Kato, Masato
AU - Lin, Yi
AU - McKnight, Steven L.
PY - 2017/3/16
Y1 - 2017/3/16
N2 - Low-complexity (LC) sequences, typically believed to be incapable of assuming structural order, are abundant constituents of the proteomes of all eukaryotic organisms. These sequences have emerged as critical components for formation of meso-scaled, sub-cellular organelles not invested by surrounding membranes, exemplified by RNA granules. We have observed that LC domains of many RNA binding proteins known to be constituents of RNA granules readily form labile cross-β polymers under physiological conditions. Several lines of experimentation have shown that formation of labile, cross-β polymers assembled from LC domain monomers is important for formation of RNA granules. Among the various experiments we have carried out, hydrogel binding assays have evolved as a versatile technique allowing a reliable means of assessing polymer formation and the binding of heterotypic cellular components integral to the formation of RNA granules. This article presents methods allowing for the production of hydrogel droplets composed of LC domain polymers. We further describe methods allowing straightforward assessment for binding of test LC domains to hydrogel droplets by fluorescence microscopy.
AB - Low-complexity (LC) sequences, typically believed to be incapable of assuming structural order, are abundant constituents of the proteomes of all eukaryotic organisms. These sequences have emerged as critical components for formation of meso-scaled, sub-cellular organelles not invested by surrounding membranes, exemplified by RNA granules. We have observed that LC domains of many RNA binding proteins known to be constituents of RNA granules readily form labile cross-β polymers under physiological conditions. Several lines of experimentation have shown that formation of labile, cross-β polymers assembled from LC domain monomers is important for formation of RNA granules. Among the various experiments we have carried out, hydrogel binding assays have evolved as a versatile technique allowing a reliable means of assessing polymer formation and the binding of heterotypic cellular components integral to the formation of RNA granules. This article presents methods allowing for the production of hydrogel droplets composed of LC domain polymers. We further describe methods allowing straightforward assessment for binding of test LC domains to hydrogel droplets by fluorescence microscopy.
UR - http://www.scopus.com/inward/record.url?scp=85021141242&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85021141242&partnerID=8YFLogxK
U2 - 10.1016/j.ymeth.2017.06.011
DO - 10.1016/j.ymeth.2017.06.011
M3 - Article
C2 - 28624540
AN - SCOPUS:85021141242
JO - Methods in Enzymology
JF - Methods in Enzymology
SN - 0076-6879
ER -