the droplet pavilion / atelier kristoffer tejlgaard
TRANSCRIPT
The Droplet Pavilion / Atelier Kristoffer Tejlgaard
Discovery of Numerous Long Noncoding RNAs
Only 2% of the human genome is used to encode proteins
(eg. FANTOM3 2005)
Long noncoding RNA (lncRNA)Dark matter
北大・生化学特別講義シンポジウム
RNAと生命現象於：農学部・総合研究棟1F・W109（多目的室） 2014年10月21日 14:00～22日 12:30
10月21日（火）14:00 RNAと生命現象（概説）：廣瀬哲郎（遺制研） 14:20 中川真一（理化学研究所）「核内ノンコーディングRNAの機能解析』15:10 伊藤秀臣（北大・理）「植物におけるsiRNAを介したトランスポゾンの制御」
ー ブレーク ー 16:00 内藤哲（北大・農） 「新生ペプチドによる翻訳制御とmRNA分解制御」16:40 稲田利文（東北大・薬）「翻訳伸長複合体の運命決定機構とその生理的意義」18:00 交流会
10月22日（水）9:30 村上洋太（北大・理） 「RNAポリメラーゼIIを介したRNAによるクロマチン構造制御」10:10 小布施力史（北大・先端生命） 「ヘテロクロマチンの機能構造と非コードRNA」
ー ブレーク ー 11:00 廣瀬哲郎（北大・遺制研） 「Architectural noncoding RNAによる細胞内構造構築」11:40 塩見春彦（慶応大・医） 「転移因子（トランスポゾン）とその抑制機構」 12:30 おわりに：内藤哲（北大・農）
協賛：文部科学省 科学研究費補助金 新学術領域研究「動的クロマチン構造と機能」（領域代表：早稲田大学 胡桃坂仁志）「新生鎖の生物学」（領域代表：東京工業大学 田口英樹）「ノンコーディングRNAネオタクソノミ」（領域代表：北海道大学 廣瀬哲郎）
企画：小布施 力史（先端生命）、村上 洋太（理学研究院）、廣瀬 哲郎（遺制研）、内藤 哲（農学院）お問い合わせ：先端生命・小布施（おぶせ） 内線 : 9015 メイル : [email protected]
このシンポジウムは、生化学特別講義の一環として開催されますが、それに関わらずご興味のある方は、どなたでも是非ご参加ください（出入り自由、事前の登録不要です）。 21 日終了後、講師の先生を囲んで交流会（会費 3500円）を開催します。参加ご希望の方は、10月 14日（火）までに小布施までメイルにてご連絡ください。
GENCODE ver 27 (Jan. 2017)15778 lncRNA genes19836 protein coding genes
Architectural Noncoding RNAs (arcRNAs)Structural scaffold of membraneless nuclear bodies
Hsr omegameiRNA HSATIIIIGS
Adaptation to hypoxia
Heat shock response
Heat shock responseMeiosis Pregnancy,
cancer progression
NEAT1
Mei2 dot w-speckle Amyloid body Nuclear stress body Paraspeckle
arcRNA
Phase separation
IDR-RBPs
~500nmYamazaki et al. Mol Cell 2018
indistinguishable (Figure S2B), suggesting that FUS LC mono-mer structure is not altered substantially by salt.
We next interrogated the dependence of full-length FUS as-sembly on salt concentration. We find that the extent of phaseseparation is not significantly affected by an increase in NaClfrom 50 mM to 150 mM but is reduced at the highest NaCl con-centration tested (300 mM) (Figure 2D). The weak salt depen-dence of full-length FUS assembly suggests that, like for FUSLC phase separation, the interactions stabilizing full-lengthFUS liquid-liquid phase separation are not primarily electro-static. However, full-length FUS assembly is not enhanced byincreasing salt as observed for FUS LC and has a lower criticalconcentration for phase separation than FUS LC. Therefore,these data suggest that interactions outside of FUS LCcontribute to phase separation of full-length FUS.
RNA Enhances Phase Separation of FUSSelf-assembled forms of FUS are thought to be nucleated byRNA binding and subsequently recruit RNA polymerase II viadirect LC domain interaction with tyrosine-containing CTD hep-tad repeats (Kwon et al., 2013; Schwartz et al., 2012). However,full-length FUS is a promiscuous RNA binder with little RNAsequence or structure preference (Wang et al., 2015) and thecontribution of FUS LC to RNA binding is unknown. Therefore,we tested the ability of nonspecific eukaryotic RNA preparations(desalted solutions of torula yeast RNA) to bind to FUS LC andnucleate its assembly. Titration of up to 5mg/ml of RNA (5:1 ratioof RNA:FUS LC by weight) did not result in phase separation of50 mM FUS LC, conditions which resulted in phase separation
of the prion-like domain of the related protein TAR DNA-bindingprotein 43 (TDP-43) (data not shown). Additionally, no significantchemical shift or intensity differences in the NMR spectrum ofFUS LC could be observed along this yeast RNA titration (Fig-ure S3A). Therefore, we find no evidence for direct interactionsof RNA with monomers of FUS LC.Because RNA enhances the formation of fibrous assemblies of
full-length FUSpresumably via binding to the RRMand zinc fingerdomains andRGGmotifs (Schwartz et al., 2013), we tested if RNAcan also enhance phase separation of full-length FUS. Weobserved the greatest extent of phase separation at an RNA:FUSratio of 0.4:1 by mass (Figure 3A). The highest concentrations ofRNA led to decreased phase separation, below that of FUSwithout RNA. These results mirror those of Cech and coworkerswho observed that multiple FUS monomers can simultaneouslybind substoichiometric amounts of RNA to induce formation offibrous FUS assemblies, but that higher RNA amounts solubilizeFUS (Schwartz et al., 2013). Taken together, these results supportthe view that the RNA-dependence of FUS phase separation canbe enhanced by FUS-RNA contacts but that the RNA contactsare not primarily mediated by the LC domain (Han et al., 2012;Kato et al., 2012; Kwon et al., 2013).
The C-Terminal Domain of RNA Polymerase II NucleatesFUS LC Assembly and Partitions into the Phase-Separated State of FUS LCNext, we tested if the 26 degenerate repeats of the C-terminaldomain of the human RNA polymerase II (CTD) interact notonly with fibrillar forms of FUS LC assembled into hydrogels asdemonstrated by McKnight and coworkers but also with liquidphase-separated FUS LC. CTD expressed in Escherichia coli ishighly soluble and does not aggregate or phase separate at con-centrations we tested (up to 500 mM). Surprisingly, addition of50 mM CTD to samples of 350 mM FUS LC at 25!C in salt-freebuffer induces extensive phase separation as well as rapid ag-gregation and precipitation at conditions and concentrationswhere FUS LC and CTD alone are both monomeric and soluble.Lowering the amount of FUS LC to 50 mM equimolar concentra-tions resulted in stable liquid phase separation (droplet forma-tion) at room temperature. We confirmed this interaction usingfluorescence microscopy of CTD incorporating an N-terminalGFP fusion and observe that GFP-CTD localizes to FUS LCphase-separated states (Figure 3B). Taken together these datademonstrate that the C-terminal domain of RNA polymerase IIcan directly interact with FUS LC domain phase-separatedstates and can nucleate their assembly.To further interrogate the interaction between FUS and CTD,
we measured the effect of FUS LC phase separation on theNMR spectrum of CTD (Figure S3B) by comparing samples of50 mM 15N CTD with and without 50 mM FUS LC in salt-free20mMMES (pH 5.5) at 25!C, which spontaneously phase-sepa-rate into a suspension of micron sized droplets (see above).Except for small chemical shift differences in 3 residues (FiguresS3C andS3D), the addition of FUS LC and the subsequent phaseseparation caused no changes in the NMR spectrum of 15NCTD.However, peak intensities in two-dimensional NMR spectrashow that the CTD signal is nearly uniformly attenuated to"75% of the control sample (Figure S3E). This loss of signal
0
0.05
0.1
0.2:1
mass ratio of RNA: full-length FUS all samples at 5 µM MBP-FUS
control
(-TEV) 0:1
0.4:1
1:10.4:1 2:1 4:1Turb
idity
(600
nm
, 1.5
mm
pat
h)
B
A
FUS LC + GFP-CTD
25 μm25 μm
Figure 3. Intermolecular Interactions in Phase-Separated FUS(A) Phase separation of 5 mM FUS (after cleaving from MBP fusion by TEV
protease, see Figure 2B) is enhanced by addition of torula yeast RNA up to a
weight ratio of 0.4:1.
(B) Fusions of GFP to the 26 degenerate heptad of the C-terminal domain of
RNA polymerase II (GFP-CTD) localize to phase-separated states of FUS LC.
Data are represented as mean ± SD.
See also Figure S3.
4 Molecular Cell 60, 1–11, October 15, 2015 ª2015 Elsevier Inc.
Please cite this article in press as: Burke et al., Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA PolymeraseII, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.09.006
Liquid droplet
FUS
Liquid-liquid phase separation and phase transition induced by various multivalent interactions
Prion-like domains in paraspeckle formation t�)FOOJH�FU�BM� 535
Materials and methods
Yeast two-hybrid interaction screenThe combinatorial yeast two-hybrid interaction screen was per-formed adopting a method similar to Golemis et al. (2011)
and Vojtek et al. (1993). In brief, yeast strains L40 (genotype: MATa, his3-Δ200, trp1-901, leu2-3, 112, ade2-101, LYS2::(lex-Aop)4-HIS3, URA3::(lexAop)8-lacZ, GAL4) and AMR70 (geno-type: MATα, his3-Δ200, ade2-101, trp1-901, leu2-3, 112, gal4Δ, met-, gal80Δ, MEL1, URA3::GAL1UAS -GAL1TATA-lacZ) were
YFP-RBM14-PLD (lane 3) but not YFP protein (lane 1) or either of the RBM14 PLD mutants (lanes 4 and 5). Bottom panels show anti-GFP Western of the same blot. WB, Western blot. (f) Schematic of paraspeckle rescue experiment and graph showing that transient, overexpressed, wild-type FUS or RBM14 can rescue paraspeckles after knockdown of endogenous FUS or RBM14, whereas the vector control or the Y→S mutant cannot. **, P < 0.02; means ± SD.
Figure 4. The RBM14 PLD forms a hydrogel with amyloid-like properties. (a) Coomassie blue staining of SDS-PAGE of purified recombinant proteins with evidence of some degradation for RBM14. GFP-FUS-PLD (lane 2), GFP-RBM14-PLD (lane 3), GFP-RBM14-PLD partial Y→S (lane 4), and GFP-RBM14-PLD All Y→S (lane 5) are shown. Size markers are shown in lane 1. (b) Photos of hydrogels formed by cooled, concentrated preparations of soluble GFP-FUS-PLD (left), GFP-RBM14-PLD (middle), and GFP-RBM14-PLD partial Y→S (right). The GFP-RBM14-PLD All Y→S was incapable of forming hydrogels (bottom). Bar, 2 mm. (right) Coomassie blue staining of hydrogel material, denatured and subject to SDS-PAGE, showing that hydrogels are enriched in full-length pro-teins. (c) Representative SEM images showing the fibrillar nature of hydrogels made with GFP-FUS-PLD (left), GFP-RBM14-PLD (middle), and GFP-RBM14-PLD partial Y→S (right). Bars, 200 nm. (d) X-ray diffraction of hydrogels made with GFP-FUS-PLD (left), GFP-RBM14-PLD (middle), and GFP-RBM14-PLD partial Y→S (right), showing the typical amyloid rings at 4.6 and 10 Å. (e) SDS solubility assay showing GFP-FUS-PLD, GFP-RBM14-PLD, or GFP-RBM14-PLD partial Y→S hydrogels are soluble in 2% SDS, whereas the pathological form (Htt46Q) of Huntingtin protein is not.
on September 30, 2015
jcb.rupress.orgD
ownloaded from
Published August 17, 2015
Hydrogel
Fiber
β-amyloid
Intrinsically disordered region (IDR)
RNA
Architectural Noncoding RNAs (arcRNAs)Structural scaffold of membraneless nuclear bodies
Hsr omegameiRNA HSATIIIIGS
Adaptation to hypoxia
Heat shock response
Heat shock responseMeiosis Pregnancy,
cancer progression
NEAT1
Mei2 dot w-speckle Amyloid body Nuclear stress body Paraspeckle
arcRNA
Phase separation
IDR-RBPs
~500nmYamazaki et al. Mol Cell 2018
Common sets of factors for nuclear body formation
Paraspeckle
SWI/SNF
NONO, FUS
Yamazaki et al.Mol Cell 2018
Nuclear stress body
SWI/SNF
SRSFs, SAFB
Kawaguchi et al.PNAS 2015
ISWI
Nona, Hrb87F
Omega speckle
3488
RESULTS
hsrω nuclear transcripts are present asnucleoplasmic speckles in all cell typesWe used digoxigenin-labeled anti-sense riboprobecorresponding to the repeat region of the hsrω gene (Lakhotiaand Sharma, 1995) to hybridize in situ with the large (>10 kb)hsrω-nuclear (hsrω-n) transcripts in intact (Fig. 1h-n) orpartially squashed (Fig. 1a-g,o-t) tissues from normally grownor heat-shocked larvae and adult flies. The hybridization wasdetected using a rhodamine-conjugated anti-dig antibody. In allthe cell types examined, the largest hsrω-n transcripts wereseen as small speckles in the nucleoplasm and at one single siteon chromatin. The RNA:RNA hybridization signal on thesingle chromosomal site was always the largest. Observationson the RNA:RNA in situ hybridization in salivary glandpolytene chromosome spreads (Lakhotia and Sharma, 1995;our other unpublished observations) also showed that the onlychromosomal site where the hsrω-n transcripts were presentwas the 93D locus. Therefore, in other cell types also, thechromosomal site of hybridization of the riboprobe wasidentified as the 93D locus. In addition totheir location at the site of transcription (the93D locus), the hsrω-n transcripts were alsopresent in the nucleoplasm of all untreatedlarval and adult diploid (Fig. 1a-g) andpolytene (Fig. 1h-t) cell types as variablenumbers of discrete small speckles. We namethese novel speckles formed by the hsrωnuclear transcripts as ‘omega speckles’. Thesignal at the 93D chromosomal site includeda diffuse staining in the core regionsurrounded by speckles similar to those seenfree in the nucleoplasm. The number ofomega speckles in a nucleus varied in a cell-type-specific pattern. The small cells in larvalbrain and imaginal discs showed about 6-8
speckles/nucleus (Fig. 1a,c). On the other hand, as reportedearlier (Lakhotia et al., 1999), the somatic cyst cells of adulttestis showed nearly 100-120 omega speckles in each nucleus(Fig. 1e). The large polytene nuclei in larval salivary glands(Fig. 1h) showed the maximum number (>1000/nucleus) ofomega speckles while the polytene cells in larval gastriccaecum (not shown) and mid gut (Fig. 1k) had very fewspeckles (approx. 4-5/nucleus). The hind gut polytene nuclei,on the other hand, showed nearly 40 omega speckles pernucleus (Fig. 1m). The larval Malpighian tubule showed about80-100 speckles/nucleus (Fig. 1o). A careful examination ofDAPI-stained partly squashed polytene nuclei of larvalMalpighian tubules and certain somatic cells in adult testisrevealed that except for the one cluster of hsrω speckles thatwas on chromatin (the 93D site), all the other hsrω speckleswere present in very close proximity to the chromatin, i.e. inthe perichromatin space (see Fig. 1o,p,s,t).
It is well known that the hsrω is strongly induced by heatshock at 37°C (for a review, see Lakhotia et al., 1999). We earlierreported (Lakhotia et al., 1999) that following heat shock, thenucleoplasmic omega speckles in larval Malpighian tubules and
K. V. Prasanth and others
Fig. 1. Localization of hsrω-n RNA byfluorescence (red) in situ hybridization using dig-labeled pDRM30 riboprobe in unstressed(a,c,e,h,k,m,o,r; CON) or heat-shocked(b,d,f,g,i,j,l,n,p,q,s,t; HS) cells of larval brain (a,b;LBr), wing imaginal disks (c,d; WD), adult testiscyst cells (e-g; Cyst cell), larval salivary glands(h-j; SG), mid gut (k,l; MG), hind gut (m,n; HG),Malpighian tubule (o-q; MT) and adult testis(somatic) polytene cells (r-t; T Somatic cell).Except in a-d and h-n, DNA was counterstainedwith DAPI (blue fluorescence). In unstressed cells(a,c,e,h,k,m,o,r), in addition to a variable numberof nucleoplasmic omega speckles, a large signal isseen in each nucleus (marked by an arrowhead inh and o) on the 93D6-7 site. Heat shock(b,d,f,g,i,j,l,n,p,q,s,t) results in aggregation ofspeckles into larger clusters and on the 93D site;in many heat-shocked nuclei, the hybridization isrestricted essentially to the 93D site (g,q and t).The polytene nuclei in p,q and t, were partiallysquashed to reveal the proximity of omegaspeckles with chromatin. Arrowheads (j,p,q,t),93D locus; arrow (t), omega speckle clusters. Bars(apply to a row), 10 µm.
Lakhotia group
eg. PLos Genet 2011
arcRNA
RNA binding protein w/ IDR
Chromatin remodeler
specific
functio
nsofthese
subdomain
sare
stillpoorly
character-
ized,butat
thedescrip
tivelev
el,they
areco
nsisten
twith
phase-
separated
systems.
Cyto
plasm
icbodies
aremore
granular
inmorphology
andhav
e
functio
nsoften
relatedto
translatio
nal
contro
lan
d/ormRNA
stabil-
ity.Theprocessin
gbody(P
body)
fallsinto
this
secondcatego
ry,in
which
translatio
nis
stalledan
dtran
scripts
aretargeted
fordegrad
a-
tionbyexo
nucleases
(Park
er&Sh
eth,2007)
orselectiv
ereactiv
ation
oftran
slation(A
rribere
etal,2011).
Stressgran
ules
arerelated
toP
bodies,
inthat
they
contain
translatio
nally
repressed
mRNA,but
form
inresp
onse
toheat,
osm
otic,
and
chem
icalstress
stimuli.
Figu
re2illu
stratesch
anges
seenin
several
bodies
durin
gcellu
lar
Nucleus
Cytoplasm
Cajal bodies
Nucleoli
HLB
s
Paraspeckles
P bodies
AC
B
Speckles
Figure1.
Inhom
eostaticcellular
conditions,dynamic
fluiddroplets
demix
fromsurrounding
nucleoplasm.
(A)Nucleoli,Cajalbodies
(CBs),histonelocus
bodies(HLBs),speckles,and
paraspecklesparticipate
inRN
Aand
RNPbiogenesis
inthe
nucleus.Associatedwith
chromosom
alloci,these
nuclearbodies
containspecific
RNAs
andproteins
thatpass
inand
outofnuclear
bodiesduring
RNPassem
bly.Unstable
RNAs
concentratein
Pbodies
inthe
cytoplasm,w
heremRN
Adecay
factorsco-localize.(B)Analogous
dynamics
andfluid
propertiesare
obtainedwhen
apurified
RNA-binding
proteinwith
alow
-complexity
regionisincubated
inwith
RNAand
observedover
timeinvitro.(C)Electron
micrograph
ofadroplet
showing
overallsphericalshapewith
anirregular
outline.Micrographs
reproducedfrom
Lietal(2012).
Glossary
Many
ofthe
termsused
inthe
recentliterature
havemeanings
thatare
overlappingor
referto
subtledifferences
between
concepts.Here,
weprovide
theclassical
definitionsfor
theseterm
sand
comment
ontheir
usage.Liquid
–liquidphase
separation(LLPS)
Itisthe
phenomenon
inwhich
solutesspontaneously
separateinto
adem
ixedliquid
phasesuspended
within
thebulk
solvent.Conventionally,the
soluteisaflexible
chainpolym
er,butthe
termis
alsoapplied
tobiological
macrom
oleculesthat
may
nothave
aflexible
chain-liketertiary
structure.Low
-complexity
domain
(LCD)
Itisaregion
within
aprotein
thatcontains
anoverrepresentation
ofasubset
ofam
inoacids
inthe
primary
sequence.Often
thisoccurs
asarepeat
motif,but
repeatsare
notarequirem
ent.Intrinsically
disorderedregions
(IDRs)
Theseare
theprotein
domains,often
containinglow
-complexity
sequencesthat
appearto
lackwell-defined
secondaryand
tertiarystructure.Som
eIDRs
havebeen
determined
experimentally,w
hileothers
areinferred
andmay
bestructured
incertain
contexts.Droplet
Itisthe
sphericalfluid
morphology
adoptedby
phase-separatedmacrom
oleculesin
solution.Droplets
havemeasureable
surface
tensionand
viscosity.Molecular
constituentsdiffuse
within
themand
canexchange
with
thebulk
solvent.Hydrogel
Itisthe
hydratedmatrix
formed
bycross-linked
proteinpolym
ers.These
polymers
arebest
thoughtof
asastable
colloidalsolid
suspendedin
water.
AggregateItisasolid
formation
composed
ofproteins
thathave
precipitatedfrom
solution.Precipitationoccurs
becausewater
isexcluded
frommacrom
olecularinteractions
tothe
extentthat
theprotein
mass
isno
longerstably
suspendedAm
yloidItisaclass
ofprotein
aggregatecharacterized
byasem
i-regularstructure
formed
bythe
stackingof
bsheets
among
proteinmonom
ersin
trans.Theyare
experimentally
identifiedby
characteristicX-ray
diffractionpatterns
andstaining
with
thedye,
thioflavinT.
Prion-likedom
ainItisaprotein
regioncharacterized
bysequence
similarity
tothat
ofprototypical
yeastprion
proteinsdom
ains.Thesecan
bethought
ofas
aspecial
caseof
low-com
plexitydom
ain
TheEM
BOJournal
ª2016
TheAuthors
TheEM
BOJournal
Droplet
organelles?Edw
ardM
Courchaineet
al
2 Published online: June 29, 2016 HSRwHSATIIINEAT1
Nuclear body
Sasaki et al. PNAS 2009 Ninomiya et al. EMBO J 2019
RNA domains
Naganuma et al.EMBO J 2012
Extraction +shearingConventional extraction
Compared read numbers of each RNA specie
RNA-seq (Hi-Seq)
Lysed HeLa cells with TRI Reagent
Identification of >50 Semi-extractable RNAs
NEAT1FRMD8
Chujo et al., EMBO J 2017
seRNA
Genomewide search for arcRNAs by RNA extractability-seq
New arcRNA candidates!!
ArcRNA is a new taxon of lncRNAs
possess (Fig. 1). This approach should make it possible to predict thefunctions of unannotated lncRNAs and improve our understanding oftheir biological significance. In this special issue, we have assembledasmuch information as possible to provide “clues to lncRNA taxonomy”,including clues about their genomic organization, expression, process-ing, structure, chemical modifications, and interacting factors, as wellas their molecular and physiological functions and putative involve-ment in various diseases. Once established, the lncRNA taxonomy willgreatly facilitate the systematic understanding of lncRNA function, pav-ing the way to determining the enigmatic roles of noncoding regionswithin the genomes of a wide range of organisms.
Dr. Tetsuro Hirose is a professor in Institute for GeneticMedicine at Hokkaido University, Japan. He received hisPhD in 1995 from Nagoya University, Japan, and continuedhis research on RNA editing in plant chloroplast as an assis-tant professor in Nagoya University until 1999. On the occa-sion of joining Joan A Steitz's laboratory at Yale University,USA as a HFSP long-term fellowship post-doc, he changedhis research field from plant to mammal. In 2005, he wasappointed as a group leader inNational Institute of AdvancedIndustrial Science and Technology (AIST), Japan until joiningthe current place in 2013. His major research interest ismolecular mechanism of the action of long noncoding RNAs.
Dr. Shinichi Nakagawa received his PhD in 1998 from KyotoUniversity, Japan. After a HFSP long-term fellowship post-doc in Christine Holt's laboratory at University of Cambridge,UK from 1998 to 2000, hewas appointed as an Assistant Pro-fessor in Kyoto University, Japan from 2000 to 2002, a Re-searcher in RIKEN Center for Developmental Biology, Japanfrom 2002 to 2005, and an Initiative Researcher in RIKEN,Japan from 2005 to 2010, and an Associate Chief Scientist inRIKEN, Japan from 2010 to present. His major research inter-ests are function of nuclear longnoncoding RNAs andnuclearstructures of higher eukaryotes.
Tetsuro HiroseInstitute for GeneticMedicine, HokkaidoUniversity, Kita 15Nishi 7, Kita-ku,
Sapporo 060-0815, Japan
Shinichi NakagawaNakagawa RNA biology laboratory, RIKEN, 2-1 Hirosawa Wako, Saitama
351-0198, Japan
Fig. 1. Concept of lncRNA taxonomy.
2 Editorial
Hirose et al. CSHL Symp Quant Biol, in pressHirose et al. Wiley Interdiscip Rev RNA (2019)
Architectural RNA (arcRNA)
Possible functions of phase separated membraneless organelle
exhibit all three functions with varying degrees.At present, our understanding of condensatefunction lags behind the rapidly developing elu-cidation of molecular assembly mechanisms,underscoring the need for future work.
Reaction crucible
Chemical reaction rates depend on concentra-tions of reactants. Concentrating a specific set ofmolecules into the condensed state may facili-tate efficient cellular reactions between weak-ly interacting molecules (Fig. 7). Moreover, theliquid-like nature of many condensates allowsfor dynamic exchange of reactants and products,as exemplified by FRAP experiments that dem-onstrate typically rapid fluorescence recovery.The functional link between phase separationand increased reaction rates has been high-lighted in the multivalent SH3/PRM system.The actin nucleation promoting factor neuralWiskott-Aldrich syndrome protein (N-WASP)contains six PRMs, which can bind SH3 domainsof NCK and induce phase separation. MultivalentSH3/PRM interactions promote phase separa-tion, concentrating actin nucleation factors andresulting in local actin polymerization withindroplets (17, 135). Recent work shows that theseSH3/PRM–driven phase transitions can alsopromote signaling outputs both in an in vitroreconstituted system and in living cells (136).During T cell receptor (TCR) signaling, signal-ing components become activated by a series ofphosphorylation reactions, leading to the for-mation of micrometer- or submicrometer-sizedclusters (137). In the reconstituted system, linker
for activation of T cells (LAT), a critical adaptorprotein for TCR signaling, undergoes liquid-liquid phase separation that is highly depen-dent on the level of available multivalency.Higher multivalency also leads to stronger acti-vation of mitogen-activated protein kinase (MAPK)signaling in Jurkat T cells, supporting the idea ofcondensate-enhanced signaling.RNP bodies may similarly function to con-
centrate reactants and thereby enhance reactionrates, as has been suggested for nucleoli, Cajalbodies, and splicing speckles. It has also longbeen speculated that such phase-separated drop-lets may have served as protocellular reactioncrucibles (138, 139); recent theoretical work sug-gests that phase-separated droplets may haveeven been able to grow and divide through re-action cycles (140). The possibility for such drop-let protocells to concentrate RNA is particularlyinteresting, given the likely central role of RNAin early life. A model experimental system usedphase separation of polyethylene glycol (PEG)and dextran, which forms droplets that are ableto concentrate ribozymes, RNA enzymes similarto those that may have been key for the originof life. PEG/dextran droplets were able to speedup ribozyme reaction rates by nearly two ordersof magnitude (141).
Sequestration
Molecular condensation may function to seques-ter factors not required for cellular needs andthereby prevent any off-target effects (Fig. 7).The nucleolus functions in part as a reactioncrucible for rRNA biogenesis, but it has long
been thought to have additional functional roles,particularly in the cell cycle and stress-dependentsequestration of key signaling molecules (74).Cytoplasmic stress granules provide another richexample of sequestration, functioning as micro-compartments for concentrating stalled transla-tion complexes under cellular stress conditions.Among numerous factors enriched in stress gran-ules are components of signaling pathways, in-cluding target of rapamycin complex 1 (TORC1)(110). Sequestration of TORC1 into the stressgranule represses TORC1 signaling (124, 142),highlighting a link between the cytoplasmic com-partmentalization and cellular signaling. Thekinase DYRK3 localizes to stress granules andregulates their dissolution (124). Transient ex-pression of DYRK3 in HeLa cells leads to liquid-liquid phase separation to form a condensedliquid phase of DYRK3 in the cytoplasm. A kinase-deficient version of DYRK3, instead, forms moresolid-like aggregates, indicating that the kinaseactivity of DYRK3 can affect the material prop-erties of stress granules. The material propertiesof such condensates are intimately linked totheir molecular dynamics, which in turn can af-fect their ability to sequester relevant factors.Future work will be necessary to quantify theextent of sequestration and to shed more lighton the coupling between the tunable materialproperties of condensates and their multifacetedbiological functions.
Organizational hub
Liquid-liquid phase separation and the resultingcondensates also appear to be exploited by cellsto organize their internal space. One interestingrecent example suggests that liquid phase con-densation may play an important role in or-ganizing spindle assembly. BuGZ is a Xenopusmicrotubule-binding protein that is predicted tobe mostly disordered and was found to undergoliquid-liquid phase separation in vitro. The re-sulting droplets are capable of bundling andconcentrating tubulin and may play an analo-gous role in organizing the spindle in living cells(53). Another recent study suggests that the forcesarising from the surface tension of membrane-associated condensates contribute to endocytosisby promoting membrane invagination (143), echo-ing the paradigm of multiphase droplet struc-turing through surface tension effects.Liquid phase condensation appears to play
similar organization roles within the nucleus,whose internal organization is entirely achievedin the absence of membrane-bound subcom-partments (Fig. 7) (3). It has become abundantlyclear over the past decade that chromosomesand associated nuclear bodies are not randomlydistributed in the nucleus (144), and nuclear ar-chitecture is intimately associated with dynamicgene regulation (144, 145). Repressed genes oftencluster into large compact states known as hetero-chromatin, and recent work demonstrates thatheterochromatin in early Drosophila embryos,observed with heterochromatin protein 1a (HP1a),exhibits signatures of liquid droplets includingfusion and dynamic molecular exchange (146).
Shin et al., Science 357, eaaf4382 (2017) 22 September 2017 8 of 11
Organizational hub
Reaction crucible Sequestration
Signalingactivity
Fig. 7. Functional roles of intracellular phase transition.Three functional categories are shown bywhich intracellular condensates play a role in cell physiology. (Left) Concentrating a specific set ofmolecules can enhance biological reactions, which is further facilitated by the dynamic molecular natureof liquid phases. (Right) Sequestration of key signaling complexes into condensates can coordinateresponse to environmental stress and cell signaling. (Bottom) Condensates can also function as anorganizational hub. For example, the localization of nuclear bodies and chromosome organization areoften coupled.
RESEARCH | REVIEW
on July 3, 2018
http://science.sciencemag.org/
Dow
nloaded from
Nuclear stress bodies (nSBs)
Splicing control
Biamonti et al., 2004. Jolly et al., 2004
Nuclear Stress Body (nSB)
HSATIII arcRNA
Heat
TFs
HSf1
HSf1
Sat III repeats
nSB
nSB
Sat III repeats
A
C
B
Adjacent loci
Figure 2. Schematic illustration of the possible roles of nSBs in heat-shocked cells. nSBs are thought to play a rolein the cellular response to stress and cell protection. Three main hypotheses have been proposed which are notmutually exclusive: (A) Control of transcription and splicing activities. Upon heat-shock, sat III sequences andtranscripts are thought to play a role in the control of transcriptional and splicing activities throughsequestration of transcription and splicing factors (both represented as dots). Transient trapping of thesefactors could contribute to the shutdown or reprogramming of gene expression. It is also plausible that SatIII RNAs, by sequestering specific RNA-binding proteins into nSBs, may influence splicing decisions towardthe synthesis of molecules involved in the cell defense to stress. (B) Regeneration of heterochromatinstructure. In fission yeast, transcripts from pericentric regions play a role in the formation and maintenanceof heterochromatin. In human cells, sat III transcripts may also play a role in protecting heterochromaticpericentric regions following heat-shock, either as long RNA molecules or as small RNA molecules generatedby the RNAi machinery. (C) Transcriptional de-repression of genes located in the vicinity of nSBs throughposition effects. Loss of epigenetic repressive marks (red flags) at the 9q12 locus following heat shock couldabolish the transcriptional repression exerted by pericentric heterochromatin on the activity of promotergenes present in cis (here visualized in brown and green) or possibly in trans (not shown) throughchromatin opening and binding of transcription factors (TF).
G. Biamonti and C. Vourc’h
8 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a000695
HSF1
(GGAAU)n
Pericentromeric Satellite III repeats
Splicing control?
SAFB
SRSF1
SRSF9
Stress inducible HSATIII arcRNAs recruit specific RBPs to form nuclear stress bodies (nSBs)
42℃ 2h
37℃
Exploration of nSBs-controlled gene expression
Control HSATIII KD
HeLa cells(42℃ 2h→37℃ 1h)
Nuclear fractionation
PolyA(+) RNA preparation
RNA-sequencing
AAAA
AAAA
AAAAnSBs
AAAA
AAAA
AAAA
AAAA AAAA
AAAA AAAA
Control
HSATIII KD
nSBs promote intron-retention of pre-mRNAs
Intron (down) 533
Intron (up) 17
Exon (down) 3
Exon (up) 4
HSATIII KD
Control533 introns(434 genes)
AAAAAA
AAAAAA
nSB
Retained introns
HSA
TIII
KD/C
ontro
l(lo
g fo
ld c
hang
e)
GAPDH
CLK1
nSBs accelerates intron retention during stress recovery
HSATIII KD control
42℃ 2h +37℃ recovery
+2h1h
+-4h+ + +
2h1h+-
4h+
- -- -
mature mRNA
pre-mRNA
0
5
10
0
0.5
1
1.5
0
1
2
3
0
0.5
1
1.5
0
20
40
60
0
5
10
0
2
4
6
0
0.5
1
1.5
0
2
4
0
1
2
3
0
2
4
6
0
2
4
0
2
4
0
0.5
1
1.5
0
1
2
0
0.5
1
1.5
0
1
2
0
0.5
1
1.5
ABHD5
intron 5
DAB2
intron 9
DNAJB9
intron 2
EP400
intron 2
PFKP
intron 1
TAF1D
intron 2
TSR1
intron 5
RBM48
intron 3
STK4
intron 1
IR
Spliced
0
0.5
1
1.5
0.0
0.5
1.0
1.5
TAF1D
intron 3
0
0.5
1
1.5
2
0
5
10
15
CLK1
intron 3
A
42ºC
Rec
over
y
37ºC
42ºC
Rec
over
y
37ºC
42ºC
Rec
over
y
37ºC
42ºC
Rec
over
y
37ºC
42ºC
Rec
over
y
37ºC
42ºC
Rec
over
y
37ºC
42ºC
Rec
over
y
37ºC
42ºC
Rec
over
y
37ºC
42ºC
Rec
over
y
37ºC
42ºC
Rec
over
y
37ºC
42ºC
Rec
over
y
37ºC
Class 1 Class 2ControlDHSATIII
*** ** * *
*
* * **
* * *
controlHSATIII KD
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11
0
0.2
0.4
0.6
0.8
1
1.2
37°C 1h 2h 4h42ºC 37°C recovery
4 5 6
RN
A le
vels
Chinese hamster (CHO)
Human (HeLa)
Heterologous expression of HSATIII humanizes splicing regulation in non-primate cells
ControlHSATIII KD
37°C 1h 2h 4h42°C37°C recovery
4 5 6
RN
A le
vels
* *
0
0.2
0.4
0.6
0.8
1
1.2 P<0.0001P<0.0001
WT
Merge/DAPI
CHO
SRSF1
(hamster)
HSATIII
(human)
Heat shock-exposed
0
0.2
0.4
0.6
0.8
1
1.2
37°C 1h 2h 4h42ºC 37°C recovery
4 5 6
RN
A le
vels *
**P=0.005
P=0.0047
P=0.0002
Heterologous expression of HSATIII humanizes splicing regulation in non-primate cells
ControlHSATIII KD
37°C 1h 2h 4h42°C37°C recovery
4 5 6
RN
A le
vels
* *
0
0.2
0.4
0.6
0.8
1
1.2 P<0.0001P<0.0001
+hChr9
WT
Heat shock-exposed
Merge/DAPI
CHO
SRSF1
(hamster)
HSATIII
(human)
CHO(His9)
Chinese hamster (CHO)
Human (HeLa)
Purification of nSBs by HSATIII arcRNA ChIRP
HSATIII
GAPDH
Inp
ut
(10
0%
)
HS
AT
III A
SO
Ran
dom
AS
O
Inp
ut
(10
0%
)
NEAT1
37℃42℃ 2h
→ 37℃ １h
HS
AT
III A
SO
Ran
dom
AS
O
Chu et al., Mol Cell 2011
ChIRP
200
11697
66.2
45
31
kDa Inpu
t
rand
omH
SATI
II
Inpu
t
rand
omH
SATI
II
silver staining
ChIRP-MS analysis of nSB proteins
GO term Pep# % P-value
mRNA splicing, via spliceosome 53 37.59 3.04E-63
mRNA processing 33 23.40 2.90E-34
RNA splicing 30 21.28 9.60E-31
mRNA 3'-end processing 21 14.89 1.54E-29
termination of RNAPII transcription 22 15.60 8.55E-29
mRNA export from nucleus 23 16.31 8.30E-26
RNA export from nucleus 19 13.48 8.91E-25
negative regulation of mRNA splicing, via spliceosome
13 9.22 1.46E-20
gene expression 15 10.64 1.10E-18
mRNA splice site selection 10 7.09 2.81E-15
regulation of alternative mRNA splicing,via spliceosome
12 8.51 7.06E-15
RNA secondary structure unwinding 10 7.09 6.84E-11
RNA processing 11 7.80 6.46E-09
Collaborated with S. Adachi (AIST)
37℃
SRSF7
SRSF9
SRSF1
SAFB
HNRNPM
GO analysis of 141 HSATIII-interacting proteins42℃ 2h→ 37℃ １h
nSBs are detectable even after stress removal
1h 2h 4h
HS
AT
IIIS
AF
B
42℃ 2h
42℃ 2h → 37℃
37℃
Merg
ed
ChIRP-MS ChIRP-MS ChIRP-MS ChIRP-MS ChIRP-MS
CLK1 kinase is specifically recruited after stress removal
1h 2h 1h 4h
nSB
prot
eins
# of peptidesidentified by MS
42℃37℃ 37℃
Collaborated with S. Adachi (AIST)
CLK1
SRSFs
CLK kinase phosphorylates SRSFs in the nucleus
Highly-concentrated in nSBs
SRSFs (SR proteins)
SR
SF9
Fla
g-C
LK
1m
erg
e
37℃ 42℃ 2h
42℃ 2h↓
37℃ 1h
SRSF9
SRSF7
SRSF1
Heat stress Post-stress recovery
Heat stress reversibly controls phosphorylation of SRSFs
SRSF9
SRSF7
p
p
SRSF1p
SRSF9
SRSF7
p
p
SRSF1p
Shi and Manley, 2007, Shi et al., 2006, Shin et al., 2004
SRSF3p
SRSF2p
SRSF3
SRSF2
SRSF3p
SRSF2p
SRSF9
SRSF7
SRSF1
Heat stress Post-stress recovery
nSBs sequestrate the de-phosphorylated SRSFs under heat stress condition
SRSF9
SRSF7
p
p
SRSF1p
HSATIII arcRNA
SRSF1
SRSF7
SRSF9
SRSF1
SRSF7
SRSF9SRSF1
nSBSRSF9
SRSF7
SRSF9
SRSF7
p
p
SRSF1p
SRSF3p
SRSF2p
SRSF3
SRSF2
Does the recruited CLK1 re-phosphorylate SRSFs in nSBs during stress recovery?
SRSF1
SRSF7
SRSF9
SRSF1
SRSF7
SRSF9SRSF1
nSBSRSF9
SRSF7
CLK1
SRSF9
SRSF7
SRSF1
Heat stress Post-stress recovery
SRSF9
SRSF7
p
p
SRSF1p
HSATIII arcRNA
SRSF1
SRSF7
SRSF9
SRSF1
SRSF7
SRSF9SRSF1
nSBSRSF9
SRSF7
SRSF9
SRSF7
p
p
SRSF1p
SRSF3p
SRSF2p
SRSF3
SRSF2
Phosphorylation statesof SRSFs?
KH-CB19
Phos-tag SDS-PAGE & WB of SRSF9
Phos-tag technology
Kinoshita et al., Nat Prot 2009
Detection of phosphorylation states of SRSF using Phos-tag SDS-PAGE
- + - - +
42℃ heat 2h
37℃ recovery - - -2h
1h1h
2h- -
The anion selectivity indexes against SO42!, CH3COO!, Cl! and
R-OSO3! at 25 1C are 5.2" 103, 1.6" 104, 8.0" 105 and42" 106,
respectively. A manganese(II) homolog of Phos-tag (Mn2+–Phos-tag) can capture a phosphomonoester dianion, such as phospho-serine or phosphotyrosine, at alkaline pH values (B9) (Fig. 1a).This finding has led to the development of phosphate-affinity gelelectrophoresis for detecting shifts in the mobility of phospho-proteins in comparison with their nonphosphorylated counter-parts16–20. We used an acrylamide-pendant Mn2+–Phos-tag as anovel additive in a separating gel for normal SDS-PAGE. In aseparating gel containing co-polymerized Phos-tag, the degrees ofmigration of phosphoproteins are less than those of their nonpho-sphorylated counterparts because the tag molecules trap phospho-proteins reversibly during electrophoresis. On the basis of thisprinciple, we recently established a novel type of gel electrophoresis,Mn2+–Phos-tag SDS-PAGE, for the separation of phosphoproteinsfrom their corresponding nonphosphorylated analogs (Fig. 1b).The Mn2+–Phos-tag SDS-PAGE protocol offers the followingsignificant advantages: (i) no radioactive or chemical labels arerequired for kinase and phosphatase assays; (ii) the time coursequantitative ratio of phosphorylated to nonphosphorylated pro-teins can be determined; (iii) several phosphoprotein isotypes,depending on the phosphorylation status, can be detected asmultiple migration bands; (iv) the phosphate-binding specificityis independent of the kind of phosphorylated amino acid; (v) His-and Asp-phosphorylated proteins involved in a two-componentsignal-transduction system can be detected simultaneously in theirphosphotransfer reactions; (vi) separation of phosphoprotein iso-types having the same number of phosphate groups is possible;(vii) a downstream procedure, such as immunoblotting or MSanalysis, can be applied; and (viii) the phosphate-affinity procedureis almost identical to the normal. In general, the migration of thenonphosphorylated protein isotype in SDS-PAGE with Mn2+–Phos-tag becomes slower than that in normal SDS-PAGEwithout Mn2+–Phos-tag, possibly because of an electrostaticinteraction between cationic Mn2+–Phos-tag and anionic SDS-bound proteins16,17.
Previous and currently improved protocols of Mn2+–Phos-tagSDS-PAGEIn an earlier protocol using a general mini-slab PAGE system, theconcentrations of Mn2+–Phos-tag were between 20 and 100 mM,and the electrophoresis was carried out at a constant current of15–35 mA per gel for o2 h9,21–33. We recently found that a lowerconcentration (5 mM) of Mn2+–Phos-tag with a smaller current of5 mA per gel for 12 h can dramatically improve the separation of aphosphoprotein having a large molecular mass of 150 kDa from itsnonphosphorylated counterpart in a 5% (wt/vol) polyacrylamideslab gel34,35. However, even this procedure did not permit the
separation of large phosphoproteins with molecular masses ofmore than 200 kDa. Although a highly porous polyacrylamidegel is generally used for the separation of high-molecular-massproteins, B5% (wt/vol) of polyacrylamide is the minimum con-centration that permits handling of the gel after electrophoresis.In this protocol introduced, this problem was circumventedby homogeneous addition of 0.5% (wt/vol) agarose to a tenderSDS-PAGE gel containing 3% (wt/vol) polyacrylamide and 20 mMMn2+–Phos-tag36. The agarose gels or agarose–polyacrylamidecomposite gels are usually used for the separation of high-molecular-mass proteins37–39. The SeaKem Gold Agarose gel(Lonza, Rockland, ME, USA), especially provided for large DNAseparation, has been reported to work best among various types ofagarose or polyacryamide gels for detecting giant myofibrillarproteins, such as titin (3,000–4,000 kDa) and nebulin isoforms(600–900 kDa)38,39. The improved procedure using a SeaKemGoldAgarose–Polyacrylamide composite gel containing Mn2+–Phos-tagpermitted the separation of phosphoprotein isotypes having mole-cular masses of 200–350 kDa within 2 h. Similarly, a betterresolution and/or faster analysis was achievable with the amendedprocedure for proteins ofB150 to 180 kDa.We show a typical resultof the mobility shift detection of the multiple phosphorylationevents on epidermal growth factor receptor (B180 kDa) aftergrowth factor-dependent signaling in Supplementary Figure 1.This protocol for high-molecular-mass phosphoproteins retainsthe advantages of the phosphate-affinity SDS-PAGE methodology,as mentioned above.
Applications of the protocolWe herein describe a useful protocol that addresses the observationof differentially phosphorylated forms of high-molecular-mass pro-teins, such as mammalian target of rapamycin (mTOR, 289 kDa),ataxia telangiectasia-mutated kinase (ATM, 350 kDa) and p53-binding protein 1 (53BP1, 213 kDa), using a strategy of combiningpolyacrylamide, agarose and the phosphate-binding tag molecule,Phos-tag36. This solid protocol of the gel-based electrophoreticseparation could also assist in mapping low-abundance phosphor-ylation events on other large proteins in a cellular signal transductionand should increase the utility for detection of hierarchicalphosphorylation and dephosphorylation using the high-quality
p uorG gn ih si lbu
P eru taN 900 2
©na
ture
prot
ocol
s/
moc.erut an.w
ww//:ptth
N
NN N
N
N
NN
P
P
P
PP
P
PP
P
P
NN
N
OO
OO O O
–O –OO–
O–
O–
O–
P P
HN H
N
N
R-OPO32–
R-OPO32––Mn2+–Phos-tagMn2+–Phos-tag
Phosphorylated protein
Phosphatebinding site
Phos-tag
Nonphosphorylated protein
Phosphate-affinity SDS-PAGE gel
Polyacrylamide
R R
Mn2+ Mn2+
Mn2+ Mn2+
a
b
N
NN
N
N
Mn2+Mn2+
O–
P
Figure 1 | Phosphate-affinity Mn2+-Phos-tag SDS-PAGE for the mobility-shiftdetection of phosphoproteins. (a) Structure of the polyacrylamide-boundMn2+–Phos-tag and scheme of the reversible capturing of a phospho-monoester dianion (R-OPO3
2!) by Mn2+–Phos-tag and (b) schematicrepresentation for the principle of Mn2+–Phos-tag SDS-PAGE. The polya-crylamide-bound Mn2+–Phos-tag shows preferential trapping of thephosphorylated proteins, which is because of separation of the phospho-proteins from their nonphosphorylated counterparts. In this paper, we focuson the utility of the phosphate-affinity SDS-PAGE for the separation of largephosphoprotein isotypes with molecular masses of more than 200 kDa.
1514 | VOL.4 NO.10 | 2009 | NATURE PROTOCOLS
PROTOCOL
The anion selectivity indexes against SO42!, CH3COO!, Cl! and
R-OSO3! at 25 1C are 5.2" 103, 1.6" 104, 8.0" 105 and42" 106,
respectively. A manganese(II) homolog of Phos-tag (Mn2+–Phos-tag) can capture a phosphomonoester dianion, such as phospho-serine or phosphotyrosine, at alkaline pH values (B9) (Fig. 1a).This finding has led to the development of phosphate-affinity gelelectrophoresis for detecting shifts in the mobility of phospho-proteins in comparison with their nonphosphorylated counter-parts16–20. We used an acrylamide-pendant Mn2+–Phos-tag as anovel additive in a separating gel for normal SDS-PAGE. In aseparating gel containing co-polymerized Phos-tag, the degrees ofmigration of phosphoproteins are less than those of their nonpho-sphorylated counterparts because the tag molecules trap phospho-proteins reversibly during electrophoresis. On the basis of thisprinciple, we recently established a novel type of gel electrophoresis,Mn2+–Phos-tag SDS-PAGE, for the separation of phosphoproteinsfrom their corresponding nonphosphorylated analogs (Fig. 1b).The Mn2+–Phos-tag SDS-PAGE protocol offers the followingsignificant advantages: (i) no radioactive or chemical labels arerequired for kinase and phosphatase assays; (ii) the time coursequantitative ratio of phosphorylated to nonphosphorylated pro-teins can be determined; (iii) several phosphoprotein isotypes,depending on the phosphorylation status, can be detected asmultiple migration bands; (iv) the phosphate-binding specificityis independent of the kind of phosphorylated amino acid; (v) His-and Asp-phosphorylated proteins involved in a two-componentsignal-transduction system can be detected simultaneously in theirphosphotransfer reactions; (vi) separation of phosphoprotein iso-types having the same number of phosphate groups is possible;(vii) a downstream procedure, such as immunoblotting or MSanalysis, can be applied; and (viii) the phosphate-affinity procedureis almost identical to the normal. In general, the migration of thenonphosphorylated protein isotype in SDS-PAGE with Mn2+–Phos-tag becomes slower than that in normal SDS-PAGEwithout Mn2+–Phos-tag, possibly because of an electrostaticinteraction between cationic Mn2+–Phos-tag and anionic SDS-bound proteins16,17.
Previous and currently improved protocols of Mn2+–Phos-tagSDS-PAGEIn an earlier protocol using a general mini-slab PAGE system, theconcentrations of Mn2+–Phos-tag were between 20 and 100 mM,and the electrophoresis was carried out at a constant current of15–35 mA per gel for o2 h9,21–33. We recently found that a lowerconcentration (5 mM) of Mn2+–Phos-tag with a smaller current of5 mA per gel for 12 h can dramatically improve the separation of aphosphoprotein having a large molecular mass of 150 kDa from itsnonphosphorylated counterpart in a 5% (wt/vol) polyacrylamideslab gel34,35. However, even this procedure did not permit the
separation of large phosphoproteins with molecular masses ofmore than 200 kDa. Although a highly porous polyacrylamidegel is generally used for the separation of high-molecular-massproteins, B5% (wt/vol) of polyacrylamide is the minimum con-centration that permits handling of the gel after electrophoresis.In this protocol introduced, this problem was circumventedby homogeneous addition of 0.5% (wt/vol) agarose to a tenderSDS-PAGE gel containing 3% (wt/vol) polyacrylamide and 20 mMMn2+–Phos-tag36. The agarose gels or agarose–polyacrylamidecomposite gels are usually used for the separation of high-molecular-mass proteins37–39. The SeaKem Gold Agarose gel(Lonza, Rockland, ME, USA), especially provided for large DNAseparation, has been reported to work best among various types ofagarose or polyacryamide gels for detecting giant myofibrillarproteins, such as titin (3,000–4,000 kDa) and nebulin isoforms(600–900 kDa)38,39. The improved procedure using a SeaKemGoldAgarose–Polyacrylamide composite gel containing Mn2+–Phos-tagpermitted the separation of phosphoprotein isotypes having mole-cular masses of 200–350 kDa within 2 h. Similarly, a betterresolution and/or faster analysis was achievable with the amendedprocedure for proteins ofB150 to 180 kDa.We show a typical resultof the mobility shift detection of the multiple phosphorylationevents on epidermal growth factor receptor (B180 kDa) aftergrowth factor-dependent signaling in Supplementary Figure 1.This protocol for high-molecular-mass phosphoproteins retainsthe advantages of the phosphate-affinity SDS-PAGE methodology,as mentioned above.
Applications of the protocolWe herein describe a useful protocol that addresses the observationof differentially phosphorylated forms of high-molecular-mass pro-teins, such as mammalian target of rapamycin (mTOR, 289 kDa),ataxia telangiectasia-mutated kinase (ATM, 350 kDa) and p53-binding protein 1 (53BP1, 213 kDa), using a strategy of combiningpolyacrylamide, agarose and the phosphate-binding tag molecule,Phos-tag36. This solid protocol of the gel-based electrophoreticseparation could also assist in mapping low-abundance phosphor-ylation events on other large proteins in a cellular signal transductionand should increase the utility for detection of hierarchicalphosphorylation and dephosphorylation using the high-quality
p uorG gn ih si lbu
P eru taN 900 2
©na
ture
prot
ocol
s/
moc.erut an.w
ww//:ptth
N
NN N
N
N
NN
P
P
P
PP
P
PP
P
P
NN
N
OO
OO O O
–O –OO–
O–
O–
O–
P P
HN H
N
N
R-OPO32–
R-OPO32––Mn2+–Phos-tagMn2+–Phos-tag
Phosphorylated protein
Phosphatebinding site
Phos-tag
Nonphosphorylated protein
Phosphate-affinity SDS-PAGE gel
Polyacrylamide
R R
Mn2+ Mn2+
Mn2+ Mn2+
a
b
N
NN
N
N
Mn2+Mn2+
O–
P
Figure 1 | Phosphate-affinity Mn2+-Phos-tag SDS-PAGE for the mobility-shiftdetection of phosphoproteins. (a) Structure of the polyacrylamide-boundMn2+–Phos-tag and scheme of the reversible capturing of a phospho-monoester dianion (R-OPO3
2!) by Mn2+–Phos-tag and (b) schematicrepresentation for the principle of Mn2+–Phos-tag SDS-PAGE. The polya-crylamide-bound Mn2+–Phos-tag shows preferential trapping of thephosphorylated proteins, which is because of separation of the phospho-proteins from their nonphosphorylated counterparts. In this paper, we focuson the utility of the phosphate-affinity SDS-PAGE for the separation of largephosphoprotein isotypes with molecular masses of more than 200 kDa.
1514 | VOL.4 NO.10 | 2009 | NATURE PROTOCOLS
PROTOCOL
SRSF9
SRSF9
SRSF9
SRSF9
ppp
p p
p
HSATIIIknockdown control
HSATIII arcRNA accelerates CLK1-dependent re-phosphorylation of SRSF9
Phos-tag SDS-PAGE & WB of SRSF9
42℃ 2h +37℃ recovery
+2h1h
+-4h+ + +
2h1h+-
4h+
- - - -
HeLa cells
2h
37℃
42℃
37℃ 1h 2h 4h
SRSF9
SRSF9
SRSF9
SRSF9
ppp
p p
p
HSATIII lncRNA
SRSF1
SRSF7
SRSF9
SRSF1
SRSF7
SRSF9SRSF1
nSBSRSF9
SRSF7SRSF9
SRSF7
p
p
SRSF1p
Heat shock Post-stress recovery
SRSF9
SRSF7
p
p
SRSF1p
nSBs promote nuclear intron retention through phosphorylation of SRSF9 by CLK1
AAAAAA
533 introns(434 genes)
AAAAAA
Intron retention
SRSF5
SRSF3
SRSF2p
p
p
SRSF9
SRSF7
SRSF1
SRSF5
SRSF3
SRSF2
Ninomiya et al., EMBO J (2019)
p
p
p
p
p
CLK1
exhibit all three functions with varying degrees.At present, our understanding of condensatefunction lags behind the rapidly developing elu-cidation of molecular assembly mechanisms,underscoring the need for future work.
Reaction crucible
Chemical reaction rates depend on concentra-tions of reactants. Concentrating a specific set ofmolecules into the condensed state may facili-tate efficient cellular reactions between weak-ly interacting molecules (Fig. 7). Moreover, theliquid-like nature of many condensates allowsfor dynamic exchange of reactants and products,as exemplified by FRAP experiments that dem-onstrate typically rapid fluorescence recovery.The functional link between phase separationand increased reaction rates has been high-lighted in the multivalent SH3/PRM system.The actin nucleation promoting factor neuralWiskott-Aldrich syndrome protein (N-WASP)contains six PRMs, which can bind SH3 domainsof NCK and induce phase separation. MultivalentSH3/PRM interactions promote phase separa-tion, concentrating actin nucleation factors andresulting in local actin polymerization withindroplets (17, 135). Recent work shows that theseSH3/PRM–driven phase transitions can alsopromote signaling outputs both in an in vitroreconstituted system and in living cells (136).During T cell receptor (TCR) signaling, signal-ing components become activated by a series ofphosphorylation reactions, leading to the for-mation of micrometer- or submicrometer-sizedclusters (137). In the reconstituted system, linker
for activation of T cells (LAT), a critical adaptorprotein for TCR signaling, undergoes liquid-liquid phase separation that is highly depen-dent on the level of available multivalency.Higher multivalency also leads to stronger acti-vation of mitogen-activated protein kinase (MAPK)signaling in Jurkat T cells, supporting the idea ofcondensate-enhanced signaling.RNP bodies may similarly function to con-
centrate reactants and thereby enhance reactionrates, as has been suggested for nucleoli, Cajalbodies, and splicing speckles. It has also longbeen speculated that such phase-separated drop-lets may have served as protocellular reactioncrucibles (138, 139); recent theoretical work sug-gests that phase-separated droplets may haveeven been able to grow and divide through re-action cycles (140). The possibility for such drop-let protocells to concentrate RNA is particularlyinteresting, given the likely central role of RNAin early life. A model experimental system usedphase separation of polyethylene glycol (PEG)and dextran, which forms droplets that are ableto concentrate ribozymes, RNA enzymes similarto those that may have been key for the originof life. PEG/dextran droplets were able to speedup ribozyme reaction rates by nearly two ordersof magnitude (141).
Sequestration
Molecular condensation may function to seques-ter factors not required for cellular needs andthereby prevent any off-target effects (Fig. 7).The nucleolus functions in part as a reactioncrucible for rRNA biogenesis, but it has long
been thought to have additional functional roles,particularly in the cell cycle and stress-dependentsequestration of key signaling molecules (74).Cytoplasmic stress granules provide another richexample of sequestration, functioning as micro-compartments for concentrating stalled transla-tion complexes under cellular stress conditions.Among numerous factors enriched in stress gran-ules are components of signaling pathways, in-cluding target of rapamycin complex 1 (TORC1)(110). Sequestration of TORC1 into the stressgranule represses TORC1 signaling (124, 142),highlighting a link between the cytoplasmic com-partmentalization and cellular signaling. Thekinase DYRK3 localizes to stress granules andregulates their dissolution (124). Transient ex-pression of DYRK3 in HeLa cells leads to liquid-liquid phase separation to form a condensedliquid phase of DYRK3 in the cytoplasm. A kinase-deficient version of DYRK3, instead, forms moresolid-like aggregates, indicating that the kinaseactivity of DYRK3 can affect the material prop-erties of stress granules. The material propertiesof such condensates are intimately linked totheir molecular dynamics, which in turn can af-fect their ability to sequester relevant factors.Future work will be necessary to quantify theextent of sequestration and to shed more lighton the coupling between the tunable materialproperties of condensates and their multifacetedbiological functions.
Organizational hub
Liquid-liquid phase separation and the resultingcondensates also appear to be exploited by cellsto organize their internal space. One interestingrecent example suggests that liquid phase con-densation may play an important role in or-ganizing spindle assembly. BuGZ is a Xenopusmicrotubule-binding protein that is predicted tobe mostly disordered and was found to undergoliquid-liquid phase separation in vitro. The re-sulting droplets are capable of bundling andconcentrating tubulin and may play an analo-gous role in organizing the spindle in living cells(53). Another recent study suggests that the forcesarising from the surface tension of membrane-associated condensates contribute to endocytosisby promoting membrane invagination (143), echo-ing the paradigm of multiphase droplet struc-turing through surface tension effects.Liquid phase condensation appears to play
similar organization roles within the nucleus,whose internal organization is entirely achievedin the absence of membrane-bound subcom-partments (Fig. 7) (3). It has become abundantlyclear over the past decade that chromosomesand associated nuclear bodies are not randomlydistributed in the nucleus (144), and nuclear ar-chitecture is intimately associated with dynamicgene regulation (144, 145). Repressed genes oftencluster into large compact states known as hetero-chromatin, and recent work demonstrates thatheterochromatin in early Drosophila embryos,observed with heterochromatin protein 1a (HP1a),exhibits signatures of liquid droplets includingfusion and dynamic molecular exchange (146).
Shin et al., Science 357, eaaf4382 (2017) 22 September 2017 8 of 11
Organizational hub
Reaction crucible Sequestration
Signalingactivity
Fig. 7. Functional roles of intracellular phase transition.Three functional categories are shown bywhich intracellular condensates play a role in cell physiology. (Left) Concentrating a specific set ofmolecules can enhance biological reactions, which is further facilitated by the dynamic molecular natureof liquid phases. (Right) Sequestration of key signaling complexes into condensates can coordinateresponse to environmental stress and cell signaling. (Bottom) Condensates can also function as anorganizational hub. For example, the localization of nuclear bodies and chromosome organization areoften coupled.
RESEARCH | REVIEW
on July 3, 2018
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nloaded from
Phosphorylation of SRSFs
Splicing control
Two distinct mechanisms of splicing control through specific biochemical reactions in nSBs
nSB
Mechanism-I
Various memberaneless RNA foci caused by repeat expansion in neurological diseases
SCA31: UGGAA
HSATIII: GGAAU
