rbm15 binds to the rna transport element rte and provides ... · rbm15 binds to the rna transport...

Lindtner et al 1

RBM15 Binds to the RNA Transport Element RTE and Provides a Direct

Link to the NXF1 Export Pathway*

Susan Lindtner†, Andrei S. Zolotukhin

§, Hiroaki Uranishi

†, Jenifer Bear

§, Viraj Kulkarni

§, Sergey

Smulevitch§, Martina Samiotaki

¶, George Panayotou

¶ , Barbara K. Felber

§1 and George N.

Pavlakis†

From the †Human Retrovirus Section, and the

§Human Retrovirus Pathogenesis Section, Vaccine Branch,

Center for Cancer Research, National Cancer Institute-Frederick, Frederick, Maryland 21702-1201 and ¶B.S.R.C. Alexander Fleming, Vari 16672, Greece

Running Title: RBM15 Promotes mRNA Export

Retroviruses/retroelements provide

important tools enabling the identification and

dissection of basic steps for posttranscriptional

regulation of cellular mRNAs. The RNA

transport element RTE identified in mouse

retrotransposons is functionally equivalent to

CTE of Type D retroviruses, yet does not bind

directly to the mRNA export receptor NXF1.

Here, we report that the RNA binding motif

protein 15 (RBM15) recognizes RTE directly

and specifically in vitro, and stimulates export

and expression of RTE-containing reporter

mRNAs in vivo. Tethering of RBM15 to a

reporter mRNA showed that RBM15 acts by

promoting mRNA export from the nucleus. We

also found that RBM15 binds to NXF1 and the

two proteins cooperate in stimulating RTE-

mediated mRNA export and expression. Thus,

RBM15 is a novel mRNA export factor and is

part of the NXF1 pathway. We propose that

RTE evolved as a high-affinity RBM15 ligand

to provide a splicing-independent link to NXF1,

thereby ensuring efficient nuclear export and

expression of retrotransposon transcripts.

General mRNA export in eukaryotes is

mediated by NXF1 protein orthologues that are

conserved from yeast to humans and bind to the

export-ready mRNP, targeting them to the nuclear

pore complex (NPC)2 (1-6). NXF1 acts as part of a

stable heterodimer with its cofactor p15/NXT1 (7-

9). Splicing changes the mRNP protein

composition, allowing NXF1-p15 to bind and

export to occur, whereas the pre-mRNPs are

normally retained in the nucleus until completely

spliced (10-13). In particular, a set of proteins

known as exon junction complex (EJC) is

deposited onto mRNP as a result of splicing (14),

providing critical determinants for the subsequent

metabolic steps, including nuclear export, quality

control, cytoplasmic trafficking and translation

(15). EJC consists of a stably bound

core

composed of eIF4AIII, Y14-Magoh and

MLN51/Barentsz that serves as a platform for a

multitude of other EJC and EJC-associated factors

that are bound more transiently. EJC is thought to

commit the spliced mRNPs to nuclear export by

providing binding sites for the NXF1-p15 export

receptor. In one scenario, the EJC factor UAP56

recruits Aly/REF proteins, which bind directly to

NXF1-p15, which in turn tethers the export

substrate to the NPC (16-21). Alternatively, NXF1

may assemble with the spliced mRNP via

interactions with non-EJC factors such as SR

proteins: SRp20 and 9G8 (22,23), ASF/SF2 (24)

and U2AF (25). Thus, it appears that several

pathways lead to the binding of NXF1-p15 with

the export-ready mRNP. Upon NXF1-p15-

dependent targeting to NPC, such complexes are

translocated to the cytoplasm by a yet unknown

mechanism. NXF1 is a conserved export receptor

for cellular mRNAs (1-6). Proteins of the NXF

family can act on nuclear as well as on

cytoplasmic mRNA trafficking (26-28).

According to the current model, general mRNA

metabolism requires the acquisition of an export

signal as a result of splicing, whereas pre-mRNA

is generally retained in the nucleus due both to the

lack of active export and to factors retaining the

pre-mRNA in the nucleus. Retroviral transcripts

are a notable exception from this rule, because the

unspliced transcript encodes the Gag-pol

polyprotein and also serves as viral genomic

RNAs, and, therefore needs to be exported prior to

splicing. To overcome the general requirement of

splicing before export, simian Type D retroviruses

http://www.jbc.org/cgi/doi/10.1074/jbc.M608745200The latest version is at JBC Papers in Press. Published on September 25, 2006 as Manuscript M608745200

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Lindtner et al 2

and some retroelements utilize the constitutive

transport element (CTE) (29-33) that serves as

high-affinity NXF1 ligand (3), thereby providing

constitutive, splicing-independent export signals.

We had discovered and characterized a novel

family of cis-acting RNA transport elements

(RTE) that are present abundantly in the mouse

genome and are associated with intracisternal A-

particle retroelements (IAP) (34,35). RTE is

functionally analogous to CTE, yet structurally

unrelated, and does not bind to NXF1 with high

affinity. In this work, we report the identification

of the factor that directly promotes RTE function.

We report that the RNA binding motif protein 15

(RBM15) recognizes RTE RNA specifically in

vitro, and activates export and expression of RTE-

containing reporter mRNAs in vivo. RBM15 also

binds to NXF1, and these factors act cooperatively

in promoting RTE-mediated expression. Thus,

RBM15 is a novel component of the NXF

pathway.

MATERIALS AND METHODS

Recombinant DNA!The reporter plasmids

pNLgag, pNLCgag, or pDM138 containing RTE,

mutant RTE, CTE, or RRE (34-38), pDM128/PL

and pDM128/B (39), the CMVgag/pol plasmids

containing a polylinker or the MPMV-CTE (40),

the expression plasmids for NXF1 (3), p15/NXT1

(41), HIV-1 rev (pCMVsrev) (42), pN-NXF1 and

pN-Rev (39), luciferase (RSV-luc) (43), GFP

(pFRED25, pFRED143) (44), secreted alkaline

phosphate SEAP (45), UAP56 (46) have been

described. pNLgagRTE(IAP23L92) contains the

RTE from the active retroelement IAP92L23 (47).

CMVgag/polRTEm26 contains the RTEm26

inserted into the polylinker. HA-tagged NXF1 and

UAP56, were cloned into cDNA3 (Invitrogen).

The RBM15 cDNA expressing isoform AE+S

(Genbank accession No NP_073605) was PCR-

amplified from a cDNA library clone (EHS1001-

27516, Open Biosystems) (48,49). Quick change

mutagenesis was performed to correct the open

reading frame (insertion of C at nt 1075), and to

introduce 2 aa changes correcting aa99 cac to ctc

and aa705 aga to gga as described by Ma et al.

(48,49). RBM15 C-terminally tagged with GFP

was generated upon insertion into pFRED25 (44).

RBM15 was N-terminally tagged with GST upon

insertion of RBM15 into pGEX-6P-3 (Amersham).

RBM15 C-terminally tagged with HA was cloned

into cDNA3. RBM15 C-terminally FLAG-tagged

was cloned into p3XFLAG-CMV-14 (Sigma). N-

tagged RBM15 proteins were generated by

replacing NXF1 cDNA in pN-TAP plasmid (39).

RBM15-S was obtained from S. Morris and the

FLAG-tag was removed. RBM15-L, and the

isoforms initiating at the AUG residue 45 were

generated by PCR. All RBM15 plasmids were

verified by sequencing.

Isolation of proteins binding to RTE RNA!

Biotinylated RTE and CTE RNA bound on

streptavidin beads (Dynal) were used as pull-down

bait (50). The immobilized RNAs were incubated

with micrococcal endonuclease treated nuclear

HeLa cell extract in buffer RBB (15 mM HEPES,

pH 7.9, 50 mM KCl, 0.1 mM EDTA, and 0.2 %

Triton X-100) with 300 mM NaCl (RBB-300)

supplemented with 4 !g/ml rRNA and 2 !g/!l

tRNA in 400 !l reactions at 30˚C for 2 hrs. The

beads were washed 6x in RBB-300 and the bound

proteins were eluted from the RNAs in 1 M NaCl,

separated on a 10% SDS-PAGE and visualized by

silver staining.

In-gel tryptic digestion, Nano-HPLC

separation and NSI-MS analysis!Protein bands

were excised from gels and digested with trypsin

as described (51,52). The peptides were separated

by nano-HPLC (LC Packings), introduced through

a nanospray ionization source to an ion-trap mass

spectrometer (LCQ-Deca, ThermoFinnigan) and

analyzed by tandem MS, as described (51,52).

Default score values used as cut-off parameters

during TurboSequest searches were: Xcorr > 1.0,

"Cn > 0.1, Sp > 500, Rsp < 10 and a peptide mass

tolerance of 1.0.

RNA transcription and gel mobility shift! 32

P-

labeled RTE and CTE RNAs (34) were prepared

as in (53) and unlabeled RNAs were synthesized

using MEGAScript-T7 (Ambion). 10 nM cold

RTE or CTE RNA was added to 10 fmol

radiolabeled RTE or CTE RNA, respectively, to

keep the RNA concentration constant for the

different probes. Binding reactions were

performed in 10 !l RBB-250 in the presence of 2

!g tRNA. After 30 min at room temperature, 1 µl

of a solution containing 0.2 mg/ml of heparin and

0.05% bromophenol blue was added to the

reactions, following 10 min at room temperature.

Samples were loaded onto a 6% (19:1


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acrylamide:bisacrylamide ratio) non-denaturing

polyacrylamide gel containing 50 mM Tris-Borate

pH 8.8, 0.5 mM EDTA. Electrophoresis was

carried out at 4˚C, and complexes were visualized

by autoradiography.

Cell culture, transfection, and microscopy!

Human 293 and 293T cells were transfected using

calcium phosphate technique. Human HeLa-

derived HLtat cells were transfected with

Superfect (Qiagen). For indirect

immunofluorescence, the fixed cells (54) were

incubated with murine anti-HA antibody

(Covance) at a 1:1000 dilution in PBS in the

presence of 0.2% BSA, followed by Alexa 594-

conjugated

anti-mouse IgG (Molecular Probes,

Eugene, OR). For the shuttling assay, transfected

HeLa cells were mixed with an excess of

untransfected cells and, after pretreatment with

cycloheximide, the cells were fused using

polyethylene glycol (54). For cotransfection

experiments, reporter plasmids were used at 1 !g,

in the absence or presence of 0.5 !g of plasmids

expressing export factors. All transfections

contained 0.2 !g GFP (pFRED143 or pFRED25)

or SEAP expression plasmids serving as internal

control for efficiency of transfection and poly-A

RNA preparation. Two days later, Gag (HIV

p24gag antigen capture assay, Zeptometrix),

SEAP (Phospha-light kit, Applied Biosystems),

CAT, and GFP fluorescence (54) were measured.

Nuclear and cytoplasmic mRNA was prepared

(25). The RNAs were analyzed on Northern blots

(42) using probes spanning the GFP or CAT

coding regions.

For RNAi experiments, 2x105 HeLa cells were

transfected with 10 mM SMART pool siRNA

(Dharmacon) targeted to RBM15

(GGACAGAGGTGATCGAGAT;

GAAGATAGAAGCTGTGTAT;GGACACCACC

CTTACTATA;GGTGATAGTTGGGCATATA)

or non-targeting siRNA control (Dharmacon)

using HiPerFect (Qiagen). One day later, the cells

were retransfected with the reporter plasmids

using Superfect (Qiagen). Culture media were

collected 48 h later, and Gag and SEAP were

measured. To control for the efficiency of RBM15

knockdowns, the cells were transfected with an

RBM15-FLAG expressing plasmid on day 1 and

then transfected with either RBM15 siRNA pools

or siRNA control pool on day 2. Cells were

harvested in RBB-400 buffer on day 4, and the

lysates were analyzed on immunoblots using

murine anti-FLAG antibody (M2, SIGMA) and

horseradish peroxidase-conjugated anti-murine

antibodies (Amersham). RNAi pool treated

untransfected cells were analyzed using anti-

RBM15 antiserum (ProteinTech Group, Inc,

Chicago) and anti-! actin antibody (Sigma). The

proteins were visualized by enhanced

chemiluminescence (ECL plus Western Blotting

Detection System, Amersham) and autography.

RT-PCR of the endogenously expressed

alternatively spliced RBM15 mRNAs was

performed on total poly-A containing mRNAs

isolated from HeLa and 293 cells using the Titan

One Tube RT-PCR Kit (Roche) and the PCR

products were sequenced.

Recombinant protein expression and protein

analysis!Human RBM15 was produced in E. coli

BL21(DE3)pLysS (Novagen) from pGEX-6P-3-

RBM15. Recombinant soluble GST-RBM15

protein was isolated after freezing the bacterial

pellets in PBS supplemented with 160 mM NaCl

and protease inhibitor (Pi, Roche) at -70˚C for 30

min. The lysates were treated with DNaseI

(Roche) and cleared by centrifugation.

Glutathione-agarose beads (Roche) were added

and the mixture was rotated for 1 hr at 4˚C. The

beads were washed three times and the

recombinant protein was eluted using a standard

glutathione-containing buffer. Protein

concentrations were estimated on Coomassie Blue

stained SDS-polyacrylamide gels.

RNA export from Xenopus oocyte nuclei!The

preparation of capped RTE and CTE-containing

adenovirus precursor RNA, U1"Sm RNA and

U6"ss RNA, unlabeled RTE, CTE and CTEm36

RNAs and oocyte nuclei microinjections were

described (34,55,56). RNA was extracted from a

pool of 5 oocytes after proteinase K digestion, and

equivalents of one-half oocyte were analyzed on

10% polyacrylamide gels containing 7 M urea.

In vitro protein binding assays! Metabolically

labeled reticulocyte-produced proteins were

synthesized in a coupled transcription/translation

system (TNT T7 Coupled Reticulocyte Lysate

System, Promega) and used in binding reactions

containing ~0.5 !g E. coli-produced GST-tagged

proteins (27). The binding was performed in 200

!l RBB-400 buffer in the presence of 100 !g

RNaseA. Following incubation for 15 min at room


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temperature, the beads were washed three times

with binding buffer. The bound proteins were

eluted by boiling in sample buffer, separated by

SDS-PAGE and detected by autoradiography.

Immunoprecipitation assays!Complexes of

epitope-tagged proteins

were immunopurified

from transiently transfected 293 cells. Typically,

~3x106 cells were extracted with 500 !l

of RBB-

400 buffer. The

extracts were cleared by

centrifugation at 10,000x g for 15 min at 4°C.

Immunoprecipitations were performed at 4°C for

30 min in 200 µl of RBB-400 buffer in the absence

or presence of RNase A using anti-FLAG M2-

agarose (Sigma). The precipitates were washed 6

times in RBB-400 buffer supplemented with 2M

UREA and were analyzed on Western

immunoblots using

horseradish peroxidase-

conjugated HA-antibodies (Roche), and the

proteins were visualized by enhanced

chemiluminescence (ECL plus Western Blotting

Detection System, Amersham) and autography.

RESULTS

Identification of RMB15 as an RTE binding

factor!Immobilized, biotinylated RTE RNA was

used to identify putative binding factors from

HeLa nuclear extracts in pull-down experiments.

CTE RNA was included as control in a parallel

experiment (Fig. 1A). These binding conditions

enabled the specific pull-down of NXF1 with CTE

RNA but not with inactive mutant CTEm36 RNA,

lacking the NXF1 binding sites (3,30), confirming

the binding specificity (data not shown). Four

candidate RTE-specific binding factors were

identified by microsequencing (Fig. 1A), which

are RNA helicase A (SwissProt Accession

Number: O70133), RNA binding motif protein 15

(RBM15; Q96T37), heterogeneous nuclear

ribonucleoprotein G (hnRNP G; P38159), and U1

small nuclear ribonucleprotein A (U1A; P09012).

An additional band at ~100 kDa could not be

identified because of contamination with bovine

serum albumin and was not further studied.

Subsequent experiments did not support specific

interactions of RNA helicase A, hnRNP G, and

U1A with RTE RNA (data not shown). In

contrast, RBM15 showed specific and functional

interaction with RTE as detailed below. RBM15

belongs to the spen (split end) protein family and

is conserved in eukaryotes from C.elegans to

humans (57), but its function had not been

investigated previously. Characteristic of this gene

family is the presence of 3 conserved RNA

Recognition Motifs (RRM) at the N-terminus and

the SPOC (Spen Paralogue and Orthologue C-

terminal) domain at the C-terminus.

Preferential binding of RBM15 to RTE in

vitro!We expressed recombinant RBM15

(isoform AE+S, see Fig.2A) in bacteria and

employed electrophoresis mobility shift assays to

examine binding of RBM15 to RTE RNA (Fig.

1B). Radiolabeled RTE (left panel) or CTE (right

panel) RNAs were incubated with increasing

amounts of bacterially produced GST-tagged

RBM15. Approximately 50% binding of RTE

RNA was observed at 15 nM of RBM15 (left

panel, lane 6), whereas no complex formation with

CTE RNA was detectable (right panel, lanes 9-

15), except for a weak band detectable by using

the highest concentration of RBM15 tested (50

nM, lane 16). These data show that recombinant

RBM15 binds directly to RTE RNA, in agreement

with the finding from the pull-down assay (Fig.

1A), and demonstrate that RBM15 interacts

preferentially with RTE.

Identification of novel isoforms of RBM15!

Ma et al (48,49) reported 3 isoforms of RBM15

AE+S, S, and L, which share aa 1-954 and have

distinct C-termini due to alternative splicing the

RBM15 mRNA (Fig. 2A). The isoform used

throughout this work is RBM15 AE+S spanning

aa 1-977, which has also been used by other

investigators (48,49,58). An anti-RBM15

antiserum raised against the C terminus of RBM15

AE+S (aa 677-977) became recently available,

which detects all isoforms. Testing human 293 and

HeLa cells, we found several barely visible bands

of endogenous RBM15 migrating higher than the

major band, which migrates at ~100 kDa (Fig. 2B,

lane 1). The endogenous RBM15 is significantly

smaller than our exogenously expressed RBM15

AE+S, which migrates at ~110 kDa (lane 3). Upon

inspection of the sequence, we noted that there are

2 AUGs at residue 1 and 45, respectively. Our

cDNA expression plasmid contains the optimized

Kozak AUG sequence precluding initiation at

downstream AUG. Therefore, we generated an

expression plasmid containing 900 nt of the

authentic RBM15 5’UTR obtained from the cDNA

clone. Interestingly, we found that 2 proteins were

produced (lane 2), one weaker band comigrating


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with the RBM15 AE+S band shown in lane 3, and

one strong, shorter band, suggesting that the

second AUG at position 45 is preferentially used.

This protein is slightly larger than the major

endogenous produced RBM15 (lane 1). Since the

proteins produced in lanes 2 and 3 are based on the

isoform AE+S, they represent the longest

isoforms. This data suggests that isoforms with

different C termini (see Fig 2A) are preferentially

produced. Using semi-quantitative RT-PCR, we

confirmed the presence of all 3 forms of

alternatively spliced RBM15 mRNAs in 293 cells

and the presence of the 5’UTR. We then

generated cDNA expression plasmids for all

isoforms utilizing either the AUG initiator codon

at residue 1 or 45 as outlined in Figure 2A. Figure

2C shows that the major endogenous form of

RBM15 co-migrates with RBM15-L 45-957

initiated at the internal AUG #2. In subsequent

experiments (data not shown), we confirmed that

all isoforms function and localize to the nucleus

like RBM15 AE+S, which was the isoform used

for all our studies and is referred to as RBM15 in

this work.

RBM15 promotes expression of RTE-

containing reporter mRNAs!It has been shown

that RTE promotes nucleocytoplasmic transport of

unspliced retroviral mRNA (34,35,59). We tested

the hypothesis that RBM15 binding is important

for RTE function. Two reporter plasmids,

pDM138 (37), producing chloramphenicol acetyl

transferase (CAT, Fig. 3A), and pNLgag (36),

producing HIV-1 Gag (Fig. 3B), were used to test

RBM15 function in cotransfection experiments in

the presence or absence of exogenous RBM15.

CAT or Gag are only produced from the unspliced

mRNA transcripts, which require the presence of a

strong RNA transport element such as CTE or

RTE in cis (30,34,59,60) or the Rev-responsive

element RRE and HIV-1 Rev (36,37,61), as also

shown in Figs. 3A and 3B (open bars). The

presence of cotransfected RBM15 (black bars)

further increased CAT expression by 6-fold from

plasmid DM138-RTE, but did not affect

expression from pDM138 lacking RTE (Fig. 3A).

Cotransfection of RBM15 also increased

expression of gag from pNLGag containing RTE

(~27-fold), when compared to the expression

obtained by the same plasmid in the absence of

exogenous RBM15 (Fig. 3B). RBM15 also

activated the RTE-containing pNLCgagRTE (36)

to similar extent like pNLgagRTE (Fig. 3C).

Plasmid pNLCgagRTE lacks the splice donor site

located 5’ to gag, and produces only unspliced gag

mRNA. These data indicate that RBM15 acts

independent of splicing.

Different control gag plasmids were tested to

study the specificity of RBM15 activation. No

RBM15-induced stimulation was observed using

the parent plasmid without RTE (Fig.3 A-C), or

the RRE-containing pNLgagRRE in the absence or

presence of Rev (Fig. 3B), supporting specific

action of RBM15 on RTE-containing mRNAs.

Interestingly, a gag RNA containing the CTE

transport element was reproducibly activated by

RBM15 (~4.5-fold, Fig 3B). Activation of CTE-

containing transcripts suggested that, although

RBM15 acts preferentially on the RTE-containing

RNA, it may have an additional general role in

mRNA metabolism and may participate in NXF1-

mediated export (see below).

RBM15 function requires the presence of an

active RTE!A series of characterized RTE

mutants (35), previously tested for their ability to

activate gag expression (Fig. 3D), was examined

in cotransfection experiments with the RBM15

expressing vector. The mutants used and their

ability to induce Gag expression from pNLgag are

shown in Fig. 3D. Cotransfection of RBM15

stimulated only the active RTE mutants m20, m21

m24 and m26. RBM15 did not activate the

inactive mutants m25 and m27. Thus, the ability of

exogenous RBM15 to further activate RTE-

containing mRNA correlated with the activity of

RTE. The data presented in Figs. 1 and 3

demonstrate that RBM15 specifically recognizes

RTE RNA both in vitro and in vivo, and that

exogenous RBM15 promotes increased expression

of RTE-containing mRNAs.

Functional knockdown of RBM15 by siRNA

inhibits RTE function!Since we found that the

exogenously expressed RBM15 stimulated the

mRNA expression via RTE, we further

investigated the effects of RBM15 depletion,

performing functional knockdowns with siRNA.

We first tested whether the selected pool of

siRNAs was able to reduce cotransfected RBM15

levels. Transfection of a pool of four siRNAs

targeting RBM15 led to a significant reduction of

cotransfected FLAG-tagged RBM15 protein levels

by ~75% (Fig. 4A). No off-target effects were

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and secreted alkaline phosphatase (SEAP)

transcripts, confirming the specificity of RBM15

knockdowns. Using an RBM15-specific

antiserum, we further confirmed that the

endogenous levels of RBM15 were also

specifically and efficiently downregulated by

siRNA (Fig 4B).

We next cotransfected HeLa cells with gag

expression vectors containing either RTE or CTE

together with a pool of siRNAs targeting the

endogenous RBM15 or a pool of non-targeting

siRNA. The production of secreted Gag protein

was measured in the culture supernatants. Fig. 4C

shows that RBM15-targeted siRNA significantly

(by 74%) inhibited RTE-mediated gag expression.

We also noticed an inhibitory effect, although to a

lesser extent, on the expression of the CTE-

containing gag mRNA (by 55%). This effect was

expected, because we had observed (see Fig. 3B)

that exogenous RBM15 activated CTE-containing

gag mRNA expression. Cotransfection of SEAP

plasmid, revealed only a small effect of the

RBM15-specific siRNA on the expression of the

SEAP transcript (~6% inhibition). These data

provide another line of evidence that endogenous

RBM15 is involved in RTE function. In addition,

these findings further support our hypothesis that

RBM15 participates in the NXF1 pathway, as

evidenced by its effects on CTE-containing gag

mRNAs.

RBM15 directly stimulates nuclear export of

mRNA!We examined whether RBM15 is able to

stimulate mRNA export directly by tethering

RBM15 to a CAT reporter mRNA that is normally

retained in the nucleus (25,39). The tethering

assay is based on the interaction of the RNA-

binding amino-terminal domain of # phage

antiterminator protein N (N) with its RNA binding

motif (boxB) (39,62). CAT plasmids containing

the boxB RNA binding motifs (pDM128/B; Fig.

5A) or lacking the element (pDM128/PL) were

cotransfected with plasmids expressing the factors

of interest that were fused to the # phage N-

peptide. N-protein fusions to the full-length

RBM15, or to the regions spanning aa 1-530 and

530-977, respectively, were shown to localize in

the nucleus (Fig. 5B), indicating the presence of 2

independent nuclear localization signals (NLS) in

RBM15. Using this tethering assay, we verified

(Fig. 5C) that cotransfection of plasmids

producing the N-peptide fusion to known mRNA

export factors such as HIV-1 Rev and NXF1

promoted CAT expression (black bars), whereas

no expression was found using a reporter mRNA

lacking the RNA binding elements (pDM128/PL,

open bars), in agreement with the data previously

reported by Wiegand et al (39). Importantly,

cotransfection of the N-RBM15 fusion protein also

strongly promoted CAT expression (Fig. 5C).

RBM15’s export function depended on binding to

the mRNA via the boxB elements, since it did not

activate expression of the parent DM128/PL cat

mRNA, lacking these elements (open bars). No

increase in CAT expression from pDM128/B was

found upon cotransfection of the RBM15

expression plasmid lacking the N-peptide (data not

shown). Taken together, these data demonstrate

that the observed stimulation by N-RBM15 (Fig.

5C) required direct interaction with the cat

reporter mRNA. Testing the N- and C-terminal

portions (RBM15 aa 1-530 and 530-977,

respectively), both localizing to the nucleus (Fig.

5B), revealed that RBM15’s export activity lies

entirely within its C-terminal portion (Fig. 5C).

To verify that RBM15 acts to increase the

nuclear export of cat mRNA, we analyzed the

effects of RBM15 tethering on nucleocytoplasmic

distribution of boxB-containing cat transcripts by

Northern blots (Fig. 5D). As expected, we found

that in the absence of tethered export factors the

unspliced cat mRNA was retained in the nucleus,

while the spliced transcript was efficiently

exported to the cytoplasm (Fig. 5D), in agreement

with Zolotukhin et al (25). Thus, the ratio of

unspliced (U, CAT-producing) to spliced (S, non-

protein producing) cat mRNA in the cytoplasm

can be used as a quantitative measure of export

efficiency for unspliced cat transcript. The ratios

of unspliced to spliced cat mRNA in the nuclear

fractions were not affected by any of the N-fusion

export factors, confirming that these proteins act

specifically at the nuclear export step. We found

that tethering of Rev, NXF1, or RBM15 led to an

increase in the ratio of unspliced to spliced

mRNAs in the cytoplasm. This is in agreement

with the reported properties of Rev and NXF1, the

export factors for HIV RRE- and CTE-containing

mRNAs, respectively, which increase the steady-

state levels of cytoplasmic unspliced HIV mRNAs

(29-33,36)(63,64). Together, these data provide

direct evidence that RBM15 is a bona fide mRNA


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export factor with an effector domain located in its

C-terminal portion.

RTE utilizes the NXF1 pathway for nuclear

export in Xenopus oocytes!To study the

mechanism of RTE-mediated nuclear export, we

employed an in vivo RNA export competition

assay using Xenopus laevis oocyte nuclei

microinjected with radiolabeled adenovirus-

derived pre-mRNA in the presence of increasing

amounts of unlabeled competitor RNA (Fig. 6).

Adenovirus-derived intron-lariats are efficiently

exported from the nucleus (N) to the cytoplasm

(C) only if they contain an active RNA export

element such as RTE (34,35) or CTE (56,65),

whereas the export of the spliced mRNA is not

affected, as outlined in Fig. 6D. Coinjection of

U1"Sm RNA and U6"ss RNA served as quality

controls and demonstrated proper function of the

nuclear export machinery and intactness of the

nuclei, respectively. This assay was previously

used to show that export of the CTE-containing

lariats utilizes the NXF1 pathway (3).

Using RTE RNA as competitor, we found

interference with the nuclear export of the RTE-

lariat at ~0.6 pmol of competitor (Fig. 6A, lane 3

versus lane 2 compared to lane 6 versus 5),

indicating that the RTE export pathway is

saturable. The saturating dose (~0.6 pmol RTE

RNA) was comparable to those previously

reported for the nuclear export pathways utilized

by U1 snRNP (0.5 pmol) and mRNA (0.1 pmol)

(55). The RTE competitor did not affect the export

of U1"Sm RNA (representative of U1 snRNP

export pathway). At 1.2 pmol of RTE competitor,

we found some interference with splicing,

resulting in reduced levels of intron-lariat and

increased pre-mRNA levels (lane 1 compared to

lane 7). Interestingly, the RTE competitor also

strongly inhibited the export of spliced mRNA

(see also Fig 6C), as it was previously observed

using the CTE as competitor in a similar assay

(56). Thus, RTE is exported via a saturable

pathway that overlaps with that of mRNA.

We next asked whether NXF1 was required for

RTE-lariat export, despite the lack of a high

affinity binding of NXF1 to RTE RNA (34). CTE

RNA serves as a tool to inhibit NXF1 function,

since NXF1 activity is specifically out-competed

upon coinjection of very low amounts of CTE

RNA (3,56,65). We found that the export of RTE-

lariat was strongly inhibited by CTE competitor

RNA, even at very low doses (Fig. 6B, lanes 2

versus 1 compared to lanes 6 and 5 or lanes 4 and

3). Excess CTE competitor had no effect on the

export of U1"Sm RNA, as expected (56). As an

additional control, we used the mutant CTEm36,

which lacks the high affinity NXF1-binding sites,

but maintains the stems and the overall RNA

structure, and does not compete for CTE export

(3,30). CTE and CTEm36 RNAs allow

distinguishing NXF1 effects from other potential

interactors (i.e. RNA helicase A (66-68)). Fig. 6B

(right panel) shows that CTEm36 RNA competitor

did not affect the RTE-lariat export (lanes 8 versus

7 compared to lanes 10 versus 9). These results

demonstrate a role of NXF1 in the RTE-mediated

nuclear export and suggest a potential interaction

of RBM15 and NXF1.

In the converse experiment, we tested whether

RTE RNA could compete for the export of the

CTE-containing intron-lariat. Fig. 6C shows that

coinjection of excess RTE RNA, using doses

sufficient to compete RTE-lariat export (see

Fig.6A), had no effect on the export of the CTE-

lariat (lanes 2 and 1 compared to lanes 6 and 5).

Thus, RTE does not interfere with CTE export,

which is in agreement with its low affinity to

NXF1, as revealed by our in vitro binding studies

(34). In contrast, the CTE-lariat export could be

out-competed efficiently using even low doses of

CTE competitor (~0.06 pmol; Fig. 6C, lanes 2

versus 1 compared to lanes 10 versus 9), as

expected (56). These data show that CTE RNA

competes for the export of both the CTE-lariat

(Fig. 6C) as well as the RTE-lariat (Fig. 6B, left

panel) with similar efficiency. These data are

consistent with an essential role of NXF1 in RTE

function.

RBM15 and NXF1 bind to each other and act

cooperatively!To test whether RBM15 and

NXF1 can interact, we examined whether GST-

tagged RBM15 protein can bind to reticulocyte

produced radiolabeled proteins using an in vitro

pull-down assay (Fig. 7A). We tested for

interactions of RBM15 with the human NXF1, and

as negative controls, with luciferase or with

UAP56, a DExD/H box helicase involved in

splicing and mRNA export (20,46,69,70). NXF1,

luciferase, and UAP56 were used at the same

molar concentrations in the binding reactions. We

found that the human NXF1 bound to RBM15 in

vitro, whereas no interactions with luciferase,


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Lindtner et al 8

UAP56 or ‘empty’ GST-beads were observed.

Similarly, we found that the mouse NXF2, a

highly related mRNA export factor (27), interacted

with RBM15 (data not shown). The binding

assays were performed in the presence of RNaseA,

demonstrating that the identified interactions of

RBM15 with the NXF proteins are RNA-

independent.

We further analyzed the RBM15-NXF1

interaction in vivo using FLAG-tagged RBM15

protein and HA-tagged NXF1 in cotransfection

experiments (Fig. 7B). Cotransfection of the HA-

tagged UAP56, a nuclear protein that does not

bind RBM15 in vitro, served as specificity control

in the assay. Western blot analysis confirmed

expression of HA-tagged proteins and similar

levels of the FLAG-tagged RBM15. Co-

immunoprecipitations using anti-FLAG antiserum

confirmed the presence of HA-tagged NXF1, but

not UAP56, in the RBM15-containing complex.

RNase treatment of the cell extract did not affect

this association, demonstrating RNA-independent

interactions. Thus, both the in vitro and in vivo

experiments confirmed the interaction between

NXF1 and RBM15.

We then examined the RBM15-NXF1

interaction in more detail upon cotransfection of a

series of FLAG-tagged RBM15 deletion mutants

and HA-tagged NXF1 (Fig. 7C). The use of

RBM15 deletion mutants identified the NXF1-

interacting region within aa 530-977, whereas aa

1-530 and aa 530-750 did not associate. We

concluded that the C-terminal portion of RBM15

contains an interaction site for NXF1 (Fig. 7C), as

well as the signals necessary to promote RNA

export, as revealed by the tethering assay shown in

Fig. 5. Confirming the specificity of these assays,

we used Western blot analysis to verify expression

of HA-tagged NXF1 and of the FLAG-tagged

RBM15 proteins, whereas co-

immunoprecipitations using anti-FLAG antiserum

verified the presence of HA-tagged NXF1 in the

complex containing the intact RBM15(1-977).

Taken together, the in vitro and the in vivo data

indicate a direct interaction between NXF1 and

RBM15.

Cooperativity between RBM15 and NXF1! To

probe the functional interaction of RBM15 and

NXF1, we examined whether coexpression of

RBM15 and NXF1 affects expression of RTE-

containing gag mRNA. Human 293T cells were

transfected with the gag reporter plasmid

containing the RTE in the absence or presence of

exogenous NXF1-p15, RBM15, or a combination

of both factors. Fig. 8A shows that Gag expression

was increased in the presence of exogenous

RBM15, as expected (see also Fig. 3), as well as,

by exogenous NXF1-p15, although to a lesser

extent. Importantly, we found that coexpression of

both factors led to a further increase of Gag

production, demonstrating cooperativity between

RBM15 and NXF1. Addition of both factors had a

more than additive effect on Gag production,

suggesting a synergistic interaction of these

factors. These data are consistent with the

participation of NXF1 in RTE RNA export (Fig.

6).

We next tested whether the NXF1-RBM15

interaction also affects CTE-mediated mRNA

expression (Fig. 8B). We found that cotransfection

of pNLgagCTE with NXF1-p15 leads to elevated

Gag levels, as expected (71). Interestingly, the

presence of exogenous RBM15 also activated

CTE-mediated expression. Furthermore, the

combination of NXF1-p15 and RBM15 led to an

additional increase in CTE-mediated expression.

Similar data were obtained using another reporter

plasmid expressing the HIV gag-pol transcript

(71), which was used to measure NXF1-p15

response in transfected 293T cells (data not

shown). These data show that RBM15 participates

also in the CTE-mediated reporter gene

expression. This finding is consistent with the

observation that functional knockdown of RBM15

also affected the CTE-mediated gag expression

(Fig. 4B). Thus, these findings reveal a functional

interaction of NXF1 and RBM15 promoting RTE-

as well as CTE-containing reporter mRNA export,

supporting a model proposed in Fig. 8C (see

below).

DISCUSSION

Studies of mRNA export of retroviruses and

retroelements have led to the identification and

characterization of molecular steps important for

understanding cellular gene expression. In this

report, we show that RBM15 selectively binds to

RTE RNA, a retrotransposon-derived transport

element, with high affinity. RBM15 also

specifically binds to NXF1, a key export receptor

for cellular mRNAs. These data are consistent


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Lindtner et al 9

with a model where RBM15 links the RTE-

containing mRNA to NXF1 export pathway (Fig.

8C). Using a previously reported in vivo export

assay (39), we demonstrate that RBM15 also

increases the nucleocytoplasmic transport of

reporter mRNAs containing the boxB RNA

element when tethered to the box B-binding #

phage N-peptide (Fig. 5), similar to the export

factors HIV Rev and NXF1. In addition to its

effect on RTE-containing mRNAs, we also found

that RBM15 increases expression of the reporter

mRNAs containing CTE (Figs. 3 and 8),

supporting a role of RBM15 in NXF1 export

pathway. Interestingly, RBM15 and NXF1 act

cooperatively to promote a further increase in

expression of RTE- as well as of CTE-containing

reporter mRNAs (Fig. 8A and 8B, respectively).

These findings are in agreement with our previous

observations, where RTE and CTE, present in

close proximity on reporter mRNAs (see also Fig.

8C), were found to synergize (59). This synergy

was reduced when the RNA elements were placed

at a distance, suggesting that there is an interaction

between RTE- and CTE-binding factors. The

identification of a specific interaction of RBM15

and NXF1 suggests a possible molecular

mechanism mediating the observed synergy.

We further found that the presence of excess

RTE RNA is able to out-compete export of the

spliced mRNA from Xenopus oocyte nuclei (Fig.

6). These results are similar to those previously

obtained for NXF1 using its high-affinity target,

CTE RNA, as competitor (56). One possible

explanation is that RBM15 is involved in critical

steps of mRNA export (see Fig. 8C). Our data

suggest that RBM15 function on cellular mRNA is

inhibited by the addition of excess of its high-

affinity binding RNA (RTE). Thus, these data are

consistent with a role of RBM15 as NXF1 cofactor

and provide evidence of a role of RBM15 in

general mRNA export.

RBM15 belongs to a conserved family of

proteins present as two members in most of the

species examined, whereas there are three genes in

man and mice. The human family comprises the

SPEN protein SHARP (SMRT/HDAC1-associated

repressor protein); RBM15, also called One

Twenty-Two (OTT); and the recently identified

OTT3, also called RBM15b (72). SHARP has

transcriptional repressor function (57,73,74),

which is not shared by RBM15 or OTT3 (72).

RBM15 is expressed in many tissues (49), but no

function has been attributed to this protein. In this

report, we demonstrate that RBM15 acts at the

posttranscriptional level, particularly in mRNA

export and expression. This function of RBM15 is

clearly distinct from that reported for the related

protein SHARP, which is involved in

transcriptional suppression (57,73,74). In

agreement with Hiriart et al (72), we found no

evidence that RBM15 acts as transcriptional

repressor by testing the activity of different

promoters such as HIV-1 LTR, SV40 or CMV

(data not shown). Therefore, despite their

evolutionary relationship, RBM15 and OTT3

appear to have functions distinct from that of

SHARP. Interestingly, RBM15 has been also

found fused to MKL1 (Megakaryoblastic

Leukemia 1 protein) in a translocation involving

chromosome 1 and 22, resulting in acute

megakaryoblastic leukemia (49,58,75-77). The

fusion protein consisting of RBM15 at its N-

terminus and MKL1, a transcription factor (78) at

its C-terminus, could interact with the mRNA

export machinery. While the RBM15-MKL1

fusion protein was found to maintain the specific

transactivator function of MKL1 (78), the fusion

protein lost RBM15’s posttranscriptional activator

function, as measured by its inability to activate

RTE-mediated mRNA expression (our

unpublished data). However, it is possible that the

RBM15-MKL1 fusion protein has transdominant

suppressor function contributing to the oncogenic

properties of RBM15-MKL1. Alternatively, the

possible decrease of posttranscriptionally active

RBM15 due to MKL1 fusion could affect mRNA

regulation, potentially contributing to

leukemogenesis. Elucidation of the role of RBM15

in mRNA export provides the basis for additional

testable hypotheses on the oncogenic mechanism

of RBM15-MKL1.

While this work was finalized, another member

of the SPEN family, OTT3, was reported to bind

to the EBV early protein EB2. OTT3 was further

shown to participate in splicing regulation of !-

thalassemia mRNA, supporting its role in

posttranscriptional steps of gene expression (72).

Thus, both RBM15 and the related OTT3

participate in posttranscriptional control of gene

expression. EB2 interacts with the SPOC domains

of all 3 human SPEN family proteins SHARP,

RBM15 and OTT3 (72). However, whereas


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Lindtner et al 10

SHARP and RBM15 interact with EB2’s C-

terminal portion, OTT3 interacts with its N-

terminal portion, demonstrating distinct properties

despite the high level of homology in the SPOC

domains (72). Interestingly, it is the C-terminal

portion of RBM15 containing the SPOC domain,

which provides the interaction site with NXF1 as

well as with the EB2 (72). Although there is no

recognizable motif shared among these factors, a

more in-depth analysis may reveal common

structural requirements. Alternatively, distinct

regions within the C-terminal portion of RBM15

interact with these factors. OTT3’s function in

RTE-mediated mRNA expression and its

interaction with NXF1 are currently under

investigation.

We propose that two structurally distinct but

functionally analogous RNA export elements

(RTE and CTE) have independently evolved (Fig.

8C). RTE, present in murine intracisternal A-

particle retroelements (34,35), emerged as high-

affinity ligand for RBM15. CTE, present in the

related Type D simian retroviruses, has evolved to

bind to NXF1 (29,30,33). It is believed that high

affinity binding to export factors is essential to

promote the export of the full-length unspliced

mRNA of retroviruses or retroelements. In

contrast, the vast majority of cellular mRNAs have

to undergo splicing before export, resulting in a

splicing-dependent deposition of export factors

such as NXF1. In this report, we present evidence

that RBM15 also has a role in the general mRNA

export pathway, since RTE inhibits export of the

spliced Adenovirus mRNA from the Xenopus

oocyte nuclei (Fig. 6). RBM15 also binds directly

to the major export receptor for mRNAs, NXF1.

We therefore propose that RBM15 plays a role in

the general mRNA export pathway, by interacting

with EJC via NXF1 (Fig. 8C). The molecular

mechanism by which RBM15 participates in

general mRNA metabolism remains the subject of

further studies.

Acknowledgments - We thank E. Izaurralde, B.R.

Cullen, T.R. Reddy, M.L. Hammarskjold, D.

Rekosh, S. Morris, and I. Tretyakova for materials

and discussions; G-M Zhang and P. Roth for

technical assistance; our summer student P. Sood

and our Werner H. Kirsten Student Intern

program recipient E. Chang for their

contributions; and T. Jones for editorial

assistance.

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Mol. Cell. Biol. 23, 6597-6608

FOOTNOTES

*This research was supported by the Intramural Research Program of the NIH, National Cancer Institute

at Frederick.

1To whom correspondence should be addressed: Human Retrovirus Pathogenesis Section, Vaccine

Branch, Center for Cancer Research, NCI-Frederick, Building 535, Room 209, Frederick, MD 21702-

1201. Tel.: 301 846-5159; Fax: 301 846-7146; E-mail: [email protected].

2The abbreviations used are: NPC, nuclear pore complex; EJC, exon junction complex; CTE, constitutive

transport element; IAP, intracisternal A-particle retroelements; RBM15, RNA binding motif protein 15;

ECL, enhanced chemiluminescence; RRM, RNA Recognition Motifs; SPOC, Spen Paralogue and

Orthologue C-terminal; CAT, chloramphenicol acetyl transferase; SHARP, SMRT/HDAC1-associated

repressor protein; OTT, One Twenty-Two; MKL1, Megakaryoblastic Leukemia 1 protein; RFU, relative

fluorescence units; SD, splice donor; SA, splice acceptor; NLS, nuclear localization signal.

FIGURE LEGENDS

FIGURE 1. RBM15 binds directly to RTE RNA. (A) Identification of RTE binding proteins. RTE and

CTE RNA bound to streptavidin-beads were incubated in vitro with nuclear HeLa extract. Proteins

binding stronger to RTE RNA than CTE RNA were sequenced by nanospray mass-spectrometry. The

filled arrow indicates RBM15, the open arrows indicate non-specific interactors. The identified peptides

are shown for each protein. (B) Recombinant RBM15 binds RTE RNA in vitro. Radiolabeled RTE or

CTE RNA was used in gel mobility shift assays with recombinant GST-RBM15. The radioactive RTE


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Lindtner et al 14

and CTE RNAs were adjusted to a final concentration of 10 nM, and GST-RBM15 was used at

concentrations of 1 to 50 nM as indicated. The position of probes and the RNA-RBM15 complexes are

indicated.

FIGURE 2. Identification of novel isoforms of RBM15. (A) Schematic representation of six isoforms of

RBM15. RBM15 isoforms differ in their N terminus initiating at AUGs at residue 1 and 45 of the open

reading frame, respectively, and having 3 distinct C termini due to alternative splicing. (B) Endogenous

and exogenous expression of RBM15. Western immunoblot analysis using anti-RBM15 antiserum of

untransfected human 293 cells (lane 1) or cells transfected with RBM15 expression plasmids (lanes 2 and

3). RBM15 AE+S expression plasmids contain the 900 nt of the RBM15 5’UTR (lane 2) or the Kozak

AUG placed next to the AUG at residue 1 (lane 3). (C) Analysis of cells transfected with the indicated

RBM15 isoforms using anti-RBM15 antiserum. (-) indicates untransfected cells.

FIGURE 3. RBM15 stimulates RTE-dependent reporter gene expression. (A) RBM15 activates CAT

production from pDM138-RTE. CAT is only expressed from the unspliced mRNA containing the cat

gene embedded within the HIV-1-derived env intron. The RTE, the splice donor (SD) and splice acceptor

(SA) sites are indicated. HeLa cells were transfected in the absence (open bars) or presence (black bars)

of RBM15 expressing vector. A representative experiment is shown. The presence of RTE in

pDM138RTE promoted increased levels (~7-fold) of CAT expression compared to the parent pDM138,

as expected (34). (B) RBM15 activates RTE-dependent Gag expression. Gag is only expressed from the

unspliced mRNA containing CTE, RTE or RRE. HeLa cells were transfected with the pNLgag plasmids

carrying the indicated RNA export elements in the absence (open bars) or presence (black bars) of

RBM15. pNLgagRRE was co-transfected with 0.1 µg HIV-1 Rev expression plasmid. Mean Gag values

and standard deviations are shown. The presence of RTE, CTE, or RRE/Rev protein led to an increase in

Gag production (10-, 124-, and 76-fold, respectively) as expected from previous studies (30,34,36). The

additional fold activation by exogenous RBM15 is shown. (C) RBM15 activates expression from

unspliced Gag-reporter mRNA. pNLCgag lacks splice sites and was previously shown to produce only

unspliced mRNA (36). Transfection of the pNLCgag, or pNLCgag containing RTE or the CTE in the

absence or presence of the RBM15 expression vector was performed as described for (B). The cell

extracts were analyzed for Gag expression as above. (D) Activity of RTE mutants correlates with the

ability to respond to cotransfected RBM15. The different RTE mutants shown were reported previously

(35): RTEm27 (loop deletion; open box); m20, m25 and m26 (nt changes, light grey boxes); m21 and

m24 (compensatory nt changes, dark grey boxes). The RTE activity was measured as fold activation

when inserted into pNLgag compared to the parent plasmid, not containing RTE, and were reported by

Smulevitch et al. (35). (-) and (+) signs next to the mutants indicate their activity. HeLa cells were

transfected with pNLgag containing the different RTE mutants in the absence or presence of RBM15. The

additional fold activation is shown.

FIGURE 4. Knockdown of RBM15 by RNAi preferentially inhibits RTE activity. (A) RBM15

targeting siRNA pool specifically reduced RBM15 expression. HeLa cells were transfected with 0.5 !g

RBM15-FLAG expression plasmid together with 0.2 !g of plasmids expressing GFP and SEAP. The next

day, the cells were transfected with 10 nM of a siRNA oligo pool specific to RBM15, or with nonspecific

control siRNA. RBM15 expression was analyzed by Western immunoblot. GFP and SEAP were

measured in cell lysates and supernatant, respectively. (B) Untransfected HeLa cells were treated with

siRNA pool specific to RBM15 or with non-specific control siRNA oligo pool for 2 days. Cell extracts

were analyzed on Western immunoblot using anti-RBM15 antiserum (upper panel) and anti-actin

antibody (lower panel). (C) HeLa cells were transfected with 10 nM of pools of RBM15-specific or

control siRNA oligos. One day later, the cells were re-transfected with the pNLgag plasmid containing

RTE(IAP92L23) or CTE together with a plasmid expressing SEAP. Gag expression was measured from

the culture supernatant two days later. Gag and SEAP expression in the presence of control siRNA was


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Lindtner et al 15

normalized to 100%. This experiment was performed in quadruplicate plates; means and standard

deviations are shown.

FIGURE 5. RBM15 tethered to RNA stimulates protein expression. (A) CAT is only expressed from

the unspliced mRNA containing the # phage boxB RNA binding elements (pDM128/B; (39), which

requires the presence of the N-peptide fused to an export factor such as RBM15. (B) RBM15 contains 3

conserved RNA Recognition Motifs (RRM) at the N-terminus and the SPOC (Spen Paralogue and

Orthologue C-terminal) domain at the C-terminus. HeLa cells were transfected with the plasmids

expressing the N-peptide-RBM15 (complete, 1-530, 530-977) fusion containing a C-terminal HA-tag and

were visualized by indirect immunofluorescence using the anti-HA antibody. DAPI staining was

performed to visualize nuclei. (C) Human 293 cells were transfected with 0.02 !g of either pDM128/B

containing the boxB RNA binding elements (black bars) or pDM128/PL lacking the insert (open bars)

together with 0.3 !g of plasmids expressing the indicated N-fusion proteins and CAT expression was

measured. A typical experiment is shown. (D) Northern blots were performed on nuclear and cytoplasmic

polyA RNA purified from human 293 cells transfected with 0.1 !g pDM128/B plasmid alone (-), or

together with plasmids expressing N-Rev (0.3 !g), N-NXF1 and p15 (0.3 and 0.15 !g, respectively) or N-

RBM15 (530-977) (0.3 !g). As control, all transfections included 0.1 !g of GFP expression plasmid. The

same blot was subsequently hybridized to CAT and GFP probes, as indicated. The positions of unspliced

and spliced CAT transcripts are indicated with arrowheads. Quantifications were performed using

phosphoimager analysis. Percentage of unspliced CAT mRNA (P) equals U/(U+S), where U and S are

signals of the unspliced and spliced CAT transcripts, respectively. The cytoplasmic to nuclear ratio of the

unspliced CAT mRNA is calculated. The stimulation of unspliced CAT mRNA export was normalized to

that the level obtained in the absence of co-expressed N-fusion tagged proteins. Similar data were

obtained in several independent experiments.

FIGURE 6. NXF1 is involved in RTE RNA export. Radiolabeled adenovirus-derived precursor mRNA

containing either RTE (A, B) or CTE (C) were injected into Xenopus laevis oocyte nuclei. Increasing

amounts of competitor RTE (A, C), CTE (B, C) or inactive mutant CTEm36 (B) RNAs were coinjected

as indicated. The injected RNA mixture also contained U1#Sm RNA that is exported to the cytoplasm (an

indicator of RNA export) and U6#ss, that remains in the nucleus (an indicator of nuclear integrity).

Cytoplasmic (C) and nuclear (N) fractions were prepared after 3 hr incubation. In panel A, total RNA (T)

is also shown. The positions of the precursor mRNAs, spliced mRNAs and the intron-lariat loop

containing RTE (open triangle) or CTE (filled triangle) are indicated. Asterisks indicate an aberrant RNA

species. Panel D shows a cartoon of the RNA products generated upon splicing of the adenovirus-derived

precursor mRNA (Ad) in the absence or presence of the RTE (or CTE) inserted into the intron. The intron

lariat gets only exported to the cytoplasm in the presence of RTE (or CTE), while the spliced mRNA gets

always exported.

FIGURE 7. RBM15 binds to NXF1. (A) RBM15 binds NXF1 in vitro. Bacterially produced GST-

RBM15 protein (right panel) or GST alone (left panel) were bound to beads and incubated with

radiolabeled reticulocyte-produced human NXF1, luciferase, or human UAP56. The bound (B) and 1% of

the unbound (U) fractions are shown after electrophoresis and autoradiography. (B) RBM15 binds to

NXF1 in vivo. Human 293 cells were transfected with HA-tagged NXF1 and UAP56 plasmids (3 !g and

0.2 !g, respectively) in the presence of 2 !g of the FLAG-tagged RBM15 expressing plasmid. Co-

immunoprecipitated proteins using anti-FLAG beads were probed with an anti-HA antibody after

electrophoresis (upper panel). Loads (1%) of cell extracts expressing HA-tagged NXF1 and UAP56

proteins (middle panel) and FLAG-tagged RBM15 mutants (lower panel) are shown. (C) NXF1 binds to

the C-terminal portion of RBM15 in vivo. Human 293 cells were transfected with 3 !g HA-tagged NXF1

plasmid in the absence (-) or presence of 2 !g of the indicated FLAG-tagged RBM15 expressing

plasmids. Co-immunoprecipitated proteins using anti-FLAG beads were probed with an anti-HA antibody

after electrophoresis (upper panel). Loads (1%) of cell extracts expressing HA-tagged NXF1 (middle


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Lindtner et al 16

panel) and FLAG-tagged RBM15 mutants (lower panel) are shown. Arrows mark the expected sizes of

the RBM15 mutants.

FIGURE 8. RBM15 and NXF1 act cooperatively. (A, B) Human 293T cells were transfected with 0.5

!g pNLgagRTE (A) or pNLgagCTE (B) either alone or together with 0.5 !g NXF1 and 0.1 !g p15/NXT1

or 0.5 !g of RBM15 expressing plasmids alone or a combination. 0.1 !g pBstat, an HIV-1 Tat expressing

plasmid necessary to activate expression from the LTR promoter was also included. Typical experiments

performed in triplicates (A) or duplicates (B) are shown. The mean Gag measurements and standard

deviations (A) or standard errors (B) are shown. For the transfections, the mean GFP values in panel (A)

were 15770, 20971, 17420, and 16109 relative fluorescence units (RFU) and in panel (B) 25902, 21990,

24972 and 17694 RFU, respectively. (C) Models for RBM15 participation in mRNA transport: RBM15

directly binds to the RTE RNA. NXF1 binds to the C-terminal portion of RBM15 and the NXF1-p15

heterodimer provides the signal for interaction with the NPC, thus RBM15 tethers the RTE-containing

mRNAs to the NXF1 export pathway. Smulevitch et al. (59) reported that the presence of RTE and CTE

on a reporter mRNA synergistically increased reporter gene expression. The presence of the two RNA

binding sites in close proximity may facilitate efficient interaction of RBM15 and NXF1, resulting in

increased reporter mRNA expression. RBM15 also acts as a NXF1 cofactor and, thereby, RBM15

interacts indirectly with CTE. Thus, the RBM15-NXF1 interaction with the RTE RNA or the CTE RNA

allow for export and expression of the unspliced mRNAs. RBM15 is thought to interact via NXF1 and the

components of EJC with the cellular mRNA, suggesting a role in general export of spliced mRNA.


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Figure 1

B

RBM15 (nM)

probe

complex

RNA probes: RTE CTE

1 2 3 4 5 6 7 8 9 10

0 1 2 5 10 15 25 50

11 12 13 14 15 16

0 1 2 5 10 15 25 50

A KMTPSYEIR

EKIQGEYKAAECNIVVTQPR

PSAAGINLMIGSTR

EEQRK

GERSKK

FENLDMSHRAK

GDRDLPSSR

KEDRSDGSAPSTSTASSK

GGHMDDGGYSMNFNMSSSR

ALEAVFGKIVEVLLMK

DYGHSSSR

LFIGGLNTETNEK

SMQGFPFYDKPMRGQAFVIFK

AVQGGGATPVVGAVQGPVPGMPPMTQAPR

GQAFVIFK

HDIAFVEFDNEVQAGAAR

U1A

RBM15

RNA

helicase A

hnRNP G

RTE CTE

160 KDa

105

75

50

35


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B

C5’ UTR

S+AE/ 5’UTRCMV

S+A

E/ 5

’UTR

S+A

E/

Koza

k A

UG

100 kDa

-

!-RBM15

RBM15 S+AE

1

RRM1 RRM2 RRM3 SPOC

977

AUG #1

AUG #2

45

1 969

RBM15 S

1 957

RBM15 L

45 977

RBM15 S+AE 45-977

45 957

RBM15 L 45-957

RBM15 S 45-969

96945

A954

Figure 2A-C

AUG #1

AUG #2

CMV

CCGCC

S+AE/Kozak AUG

1 2 3

100 kDa

S+A

E 1

-977

S

1-96

9

L 1

-957

L 4

5-95

7 S

45-

969

S+A

E 4

5-97

7

-

!-RBM15


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B

Ga

g e

xp

res

sio

n (

ng

p2

4)

A

%

CA

T A

cti

vit

ySD SA

CTE/RTE/RRE

LTR gagGag pA

FIGUREFIGURE 3A-B3A-B

SV40

SA

RTE

SD

pACAT

Fold activation

by RBM15:1 6 1.4 27 4.5 1.8 1.7

Fold activation

by RBM15:

none RTE none RTE CTE RRE RRE+RevRNA Export

element:

RNA Export

element:

pDM138

pNLgag

- + - + - + - - - - + + + +Cotransfection

of RBM15:

Cotransfection

of RBM15:


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CG

ag

ex

pre

ss

ion

(n

g p

24

)

1.8 32 5.3Fold activation

by RBM15:

none RTE CTERNA Export

element:

CTE/RTE

LTR gagGag pApNLCgag

- + - - + +Cotransfection

of RBM15:

FIGUREFIGURE 3C3C


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Figure 3DFigure 3D

2710RTE

1 1RTEm27

1520RTEm26

1.7 1RTEm25

6 3RTEm24

2014RTEm21

30 9RTEm20

Additional fold

activation by

cotransfected

RBM15

Fold activation of

gag reporter

mRNA

RTE

mutants

D

m26

m24

m21

m20

m25

m27

+

+

+

++

--


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Figure 4A-C

C

Inhibition by RBM15 siRNA

74% 55% 6%

% G

en

e e

xp

ressio

n

Gag reporter

RTE CTE SEAP

A

RBM15-FLAG

Control

siRNA

SEAP:

GFP:

1201 1427

2292 2191

RBM15

siRNA

Sample dilution: 1 1:2 1:4 1:5 1

B

!-RBM15

Con

trol

siR

NA

R

BM

15 s

iRNA

!-actin


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Figure 5A-C

% CAT activity

CA

1 977529

RRM1 RRM2 RRM3 SPOC

1-977

1-530

530-977

Localization of

N-RBM15 proteins

(HA-tagged)

1-530

530-977

B

boxB

elements

SASD

CAT AAA

N-R

BM

15

Export and expression

of unspliced CAT mRNA

RBM15

pDM128/B

CMV

!-HA DAPI


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N-R

BM

15

(53

0-97

7)

Cytoplasm

Unspliced >

Spliced >

Nucleus

N-R

ev

Stimulation of unspliced CAT mRNA export: 1x 1.8x 3.9x 3.5xN

-NXF1

N-R

BM

15

(53

0-97

7)

N-R

evN

-NXF1

CAT

GFP

% unspliced CAT mRNA: 74 77 82 88 12 22 52 49

Figure 5D

C/N ratio of unspliced CAT mRNA: 0.16 0.29 0.63 0.56

D by guest on January 13, 2020http://w

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AFigure 6A-D

U6"ss

pmol pmol ofof

competitor competitor

RTE

pre-mRNA

(RTE)

0.60.6 1.21.2

Ad-RTE

U1"Sm

RTE

T C N T C N T C N

spliced

*

1 2 3 4 5 6 7 8 9

pre-mRNA

(CTE)

CTE

U6"ss

U1"Sm

spliced

*

0.20.2 0.60.6 0.020.02 0.060.06

RTE CTEAd-

CTE

C N C N C N C N C N

C

1 2 3 4 5 6 7 8 9 10

pmol pmol ofof


B

U6"ss

pre-mRNA

(RTE)

U1"Sm

C N

Ad-

RTECTEm36

0.060.06 0.020.02

C N C N

0.080.08

CTE

0.040.04

Ad-

RTE

C N C N C N

spliced

*

1 2 3 4 5 6 7 8 9 10 11 12

pmol pmol ofof


RTE

D

RTE

RTE

splicing

Ad pre-mRNA Ad-RTE

intron-

lariat

spliced

Exon 1 Exon 2

intron-

lariat

spliced

Export: no yes yes yes


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Figure 7A-C

C

IP with!-FLAG

- 530-

750

530-

977

1-53

01-

977RBM15-FLAG

CoIP:

!-HA (NXF1)1% Load

75

50

35

30

105 kDA

!-FLAG

RBM15-FLAG

>

>>

>

Western:

1% Load

!-HA (NXF1)

B

NXF1-

HA

UA

P56

-HA

RBM15-FLAG

CoIP:

1% Load

1% Load

IP with!-FLAG

!-HA

!-FLAG

Western:

!-HA

75

50

75

50

AS

35 p

rote

ins

:

NXF1

Luciferase

UAP56

U B U B

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Figure 8A, B

A

5

16

44

NXF1

RBM15NXF1

+RBM15minus

Ga

g e

xp

ressio

n (

ng

)

Fold

Activation: 12 40 1101

0.4

NXF1

RBM15NXF1

+RBM15minus

Fold

Activation: 3 7 15 1

4343

21

7

2.8

B

Gag-RTE Gag-CTE

10

20

30

40

50

60

70

0

Ga

g e

xp

ressio

n (

ng

)


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NXF1

p15

Figure 8C

RBM15 is a NXF1 cofactor

RTE mRNA CTE mRNACTE mRNA

AAA

NXF1RBM15

p15

RBM15 binds RTE RNA

and interacts with NXF1

AAA

RBM15NXF1

p15

unspliced mRNA export

EJC

core

UAP56 REF

E1 E2

RBM15

p15

NXF1

AAA

Cellular mRNA

spliced mRNA export

C

RTE-CTERTE-CTE

mRNA mRNA

AAA

RBM15


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N. PavlakisSergey Smulevitch, Martina Samiotaki, George Panayotou, Barbara K. Felber and George

Susan Lindtner, Andrei S. Zolotukhin, Hiroaki Uranishi, Jenifer Bear, Viraj Kulkarni,NXF1 export pathway

RBM15 binds to the RNA transport element RTE and provides a direct link to the

published online September 25, 2006J. Biol. Chem.

10.1074/jbc.M608745200Access the most updated version of this article at doi:

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rbm15 binds to the rna transport element rte and provides ... · rbm15 binds to the rna transport...

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