Copyright 2001 by the Genetics Society of America

Positive and Negative Intronic Regulatory Elements Control Muscle-SpecificAlternative Exon Splicing of Drosophila Myosin Heavy Chain Transcripts

David M. Standiford, Wei Tao Sun, Mary Beth Davis and Charles P. Emerson, Jr.

Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennyslvania 19104

Manuscript received May 2, 2000Accepted for publication September 28, 2000

ABSTRACTAlternative splicing of Drosophila muscle myosin heavy chain (MHC) transcripts is precisely regulated

to ensure the expression of specific MHC isoforms required for the distinctive contractile activities ofphysiologically specialized muscles. We have used transgenic expression analysis in combination withmutagenesis to identify cis-regulatory sequences that are required for muscle-specific splicing of exon 11,which is encoded by five alternative exons that produce alternative “converter” domains in the MHC head.Here, we report the identification of three conserved intronic elements (CIE1, -2, and -3) that controlsplicing of exon 11e in the indirect flight muscle (IFM). Each of these CIE elements has a distinct function:CIE1 acts as a splice repressor, while CIE2 and CIE3 behave as splice enhancers. These CIE elementsfunction in combination with a nonconsensus splice donor to direct IFM-specific splicing of exon 11e.An additional cis-regulatory element that is essential in coordinating the muscle-specific splicing of otheralternative exon 11s is identified. Therefore, multiple interacting intronic and splice donor elementsestablish the muscle-specific splicing of alternative exon 11s.

THE regulated splicing of alternative exons in pre- et al. 1991), whereas the rat gene is regulated throughmRNAs is a widely utilized genetic mechanism for 59 donor competition (Chen et al. 1999). Together,

generating tissue-specific protein isoforms (Chabot these studies have demonstrated a diversity of roles1996). Muscle makes extensive use of alternative splicing played by cis-acting sequence information to provideto produce the muscle-specific contractile protein iso- either positive or negative interactions that ensure theforms in different muscle types to specialize their con- proper tissue-specific use of alternative exons.tractile properties (George et al. 1989; Nadal-Ginard These same models have also been instrumental inet al. 1991; Bandman 1992; Standiford et al. 1997). defining the factors that interact with cis-acting elementsConsequently, muscle has been useful as a model cell to mediate the proper recognition of the alternativetype to investigate molecular mechanisms that regulate exons. For instance, the cTNT ESE has been shown totissue-specific alternative exon splicing and has pro- interact with several members of the SR family of splic-vided detailed information about these processes. For ing factors (Ramchatesingh et al. 1995), while the studyinstance, molecular and biochemical studies of the of a- and b-TM transcripts has shown the involvementmechanisms that regulate the alternative splicing of the of hnRNP proteins as regulators of alternative splicingchicken cardiac troponin T gene have shown that mus- (Patton et al. 1991; Lin and Patton 1995; Perez et al.cle-specific intronic splicing enhancers function in com- 1997; Chen et al. 1999; Lou et al. 1999; Southby et al.bination with a general exonic splicing enhancer (ESE; 1999).Xu et al. 1993; Ryan and Cooper 1996) to direct the The analysis of muscle-specific alternative splicing hasinclusion of exon 5 in the embryo. Similarly, the splicing also contributed to the discovery that interactions be-of exon 2 of a-tropomyosin (a-TM) in smooth muscle tween cis-acting elements and trans-acting factors cancells requires purine-rich exonic splicing enhancers be influenced by cell or tissue-specific conditions. For(Dye et al. 1998), but the proper use of this exon also instance, polypyrimidine tract binding protein is consti-requires the repression of the default splicing of exon tutively expressed, yet can specifically antagonize the use3 (Mullen et al. 1991; Gooding et al. 1994, 1998). Stud- of a-tropomyosin alternative exon 3 in smooth muscleies of the chicken b-tropomyosin gene transcript have (Perez et al. 1997). Similar events occur in other tissuesdemonstrated a role for secondary structure in alterna- and studies have shown that splice site selection can betive exon selection (Clouet D’Orval et al. 1991; Libri differentially modified through tissue-specific phos-

phorylation of SR proteins (Du et al. 1998; Xiao andManley 1998) or through variations in the abundance

Corresponding author: Charles P. Emerson, Department of Cell and of splicing factors that act antagonistically (Gallego etDevelopmental Biology, 245 Anatomy and Chemistry Bldg., University al. 1997; Labourier et al. 1999) or have differentof Pennsylvania School of Medicine, Philadelphia, PA 19104-6058.E-mail: emersonc@mail.med.upenn.edu strengths (Stark et al. 1999), demonstrating that the

Genetics 157: 259–271 ( January 2001)

260 D. M. Standiford et al.

exact tissue-specific environment is likely to play a large pathway in its absence. Further, we find that three con-served intronic elements are essential components ofrole in directing alternative exon use. Because of this

complexity, it is unlikely that in vitro approaches to the the mechanism that directs the utilization exon 11e inthe IFM, and the in vivo analysis of these unique ele-study of alternative splicing will be sufficient to fully

replicate the conditions that exist to separate and define ments shows that they have either positive or negativeactivities that regulate the selection of exon 11e in thethe specific pattern of alternative splicing of transcripts

among different tissues. IFM. These data support a model in which there is aspecific and unique alternative splicing mechanism inWe have developed an in vivo model for studying the

mechanism that regulates muscle-specific alternative the IFM that is directed toward the inclusion of Mhcexon 11e in this muscle.splicing that is based on the analysis of Drosophila skele-

tal muscle myosin heavy chain (MHC) gene transcripts.Drosophila has a single Mhc gene consisting of 19 exons,

MATERIALS AND METHODS5 of which are represented as 2 to 5 alternatively splicedexons encoding related sequences (Figure 1A; George Constructs: The construction of the gD1048, gD1060,et al. 1989). The alternative exons in each group are gD1120, gD1168, gD1105, gD1090, gD1177, and gD1222 Mhcspliced in a mutually exclusive fashion, but the combina- minigene constructs has been described previously (Standi-

ford et al. 1997). The gD1254, gD1256, and gD1265 constructstorial use of alternative exons in different groups allows,were made using the QuickChange procedure (Stratagene,in theory, for the expression of 480 MHC isoforms.La Jolla, CA) to mutagenize the conserved intronic elementsHowever, the splicing of these exons is subject to precise (CIE) sites. Mutagenesis was conducted against an isolated

muscle-specific regulation that allows individual special- 4-kb EcoRI fragment from gD1060, which was inserted intoized muscles typically to produce only a single MHC BlueScript SK- (Stratagene) and followed the manufacturer’s

protocol. The sense and antisense mutagenic oligos for CIE1protein isoform (Hastings and Emerson 1991). Forare CCGTCCTTCTCCACGTACCAGAAATCAATTC andexample, Mhc exon 11 contains five alternatives, andGTGGAGAAGGACGGACAACGAGGATCAACG, respectively,transcripts expressed in the functionally distinct indirect and GTTTCGAACTATGGCTTAGGTGTATCTCCG and

flight muscle (IFM) and jump muscle tergal depressor CCTAAGCCATAGTTCGAAACTGGAATTGAT for the CIE2of the trochanter (TDT) differ essentially only by the mutation. Mutagenized constructs were identified by sequence

analysis and a EagI (exon 11e)-PacI (intron 12) fragment fromselection of exon 11e in the IFM and exon 11b in thethe mutant subclone was used to replace the same fragmentTDT. Exon 11 encodes the MHC “converter” domain,in gD1060 to introduce the CIE mutations. The double CIEwhich is thought to play a critical role in determining mutant was made by sequential rounds of QuickChange muta-

key functional properties of myosin (Bernstein and genesis using the same CIE1 and CIE2 mutagenic oligos. TheMilligan 1997; Dominguez et al. 1998), and the fact gD1275, gD1276, and gD1320 mutants were made by Quick-

Change mutagenesis and the same CIE1 or CIE2 mutagenicthat exon 11e is utilized only in the highly specializedoligos, but mutagenesis was done in a 1-kb EagI-KpnI fragmentIFM suggests that normal flight function is dependentfrom gD1048. These same sites were used to introduce theon the expression of this particular myosin isoform. mutagenized sequences into either gD1048 (gD1275 and

This requirement indicates that IFM-specific splicing gD1276) or gD1120 (gD1320). gD1334 was made with Quick-regulation is subject to intense evolutionary selection Change using a mutant oligo to replace the exon 11e donor

with that of exon 11b, which was done in a CIE2 mutagenizedand, for this reason, we have pursued a detailed analysisbackground. The gD1223 construct was generated by ampli-of the cis-regulatory elements that control exon 11efying Mhc nucleotides (nt) 12010–12300 (numbering fromsplicing. GenBank accession no. M61229) using oligo-containing flank-

In studies preliminary to this report, we developed ing AflIIsites (sense-GCATCACTTTAAGACCAGGTTMhc transgene reporters to investigate the regulatory GATAAGTC; antisense-GCATCACTTAAGTTTCATTTGTG

GATGC), which contain exon 11b and its native flankingsequences required for the alternative splicing of exonintronic sequence, digesting this with AflII and placing it into11 alternatives in specialized muscles (Figure 1B;the gD1060 construct that was linearized and filled at the AflIIStandiford et al. 1997). Our findings established that site. The gD1242 transgene was constructed by amplifying a

Mhc exon 11 contains sufficient cis- information to direct section from gD1048 that included exon 2 to Mhc nt 12301the normal, muscle-specific splicing of alternative ex- (intron 11b), using the exon 2 primer listed below and an

Mhc exon 11b intron antisense primer (GCCGGCTCGAGons. We also showed that nonconsensus exon 11 spliceGAAGAACCGCTTAAGCATAACG), which contains an XhoIdonors are essential for muscle-specific alternative exonrestriction site at its 59 end. A second fragment was generatedsplicing and, further, that conserved intronic elements from gD1048 using a LacZ specific antisense primer and an

located in the exon 11 interval are required components 11d intron specific primer (GCGCGCTCGAGTTTGTATTTof the alternative splicing mechanism. In this report, CATTTGTGGATGC) beginning at Mhc nt 13941, which also

contains an XhoI site. Both fragments were digested with XhoIwe have focused on the mechanism used by the IFMand were ligated to each other. This product was subjectedto direct the inclusion of alternative exon 11e into itsto an additional PCR reaction to amplify between exon 2 andprocessed transcripts. Specifically, we use directed muta-LacZ to produce a fragment that now contains an exon 11

genesis of transgenic Mhc exon 11 minigenes and RT- interval that is deleted for exon 11c through CIE3. This frag-PCR analysis to show that the IFM has a strict require- ment was digested with SacII and PacI and used to replace

the same in gD1048, resulting in the gD1242 construct. Thement for exon 11e and defaults to an exon-skipping

261Muscle-Specific Alternative Splicing in Drosophila

gD1152 gD1356, gD1357, and gD1362 constructs were made in this muscle, which conforms to the precise, IFM-by amplifying across the CIE3 and inserting this fragment in specific use of the endogenous exon 11e. Extensive mu-the appropriate background.

tational analysis on the Mhc minigene revealed that sev-Drosophila methods: All Drosophila cultures were main-eral classes of alterations could significantly disrupt thetained at 258 on standard cornmeal molasses medium (Ash-

burner 1989). Transgenic flies were generated by standard normal use of exon 11e in the IFM: (1) the removal ofmethods. The strain y1 w67c23(2) was used as the injection strain exon 11e itself, (2) the conversion of the nonconsensusand as a “wild-type” reference strain in all of the experiments. exon 11e splice donor to consensus, and (3) the removalA minimum of three independently derived transgenic lines

of a conserved intronic sequence (CIE3; Figure 1;was generated for each construct. Histochemical staining ofStandiford et al. 1997). Together, these results indicateflies for b-galactosidase activity was performed as described

by Ashburner (1989). that there is a IFM-specific splicing regulatory mecha-Reverse transcriptase-dependent PCR: RT-PCR methods nism that is designed to promote exon 11e splicing in

were essentially those followed in Standiford et al. (1997). this muscle.Briefly, for adult muscle, newly eclosed flies were collected

A key observation from these earlier studies was thatand then soaked for a few minutes in petri plates containingthe deletion of exon 11e from the gD1048 transgene100% ethanol at room temperature. After this treatment the

IFM, TDT, and other muscles become stiff and can be easily does not lead to default splicing of other alternativedissected away from each other. Separated IFM and TDT mus- exon 11s in the IFM nor does this deletion effect thecles were placed into 1.5-ml microfuge tubes containing 100% splicing of any other alternative exon 11 in their specificethanol on ice until 10–15 flies per genotype were dissected.

muscle types (Standiford et al. 1997). Here, we haveThe tubes containing the muscles were spun at 2000 rpm, atdefined the molecular response of the IFM-specific splic-48, and the ethanol was removed. The muscles were resus-

pended in 100 ml of H2O, and 0.5 ml of RNAzol reagent ing apparatus to the loss of exon 11e using an RT-PCR(Sigma, St. Louis) was immediately added along with 20 mg assay that shows that a truncated transcript results inof yeast transfer RNA (Sigma) as carrier. Total RNA was iso- the IFM when exon 11e is removed from the minigenelated with the RNAzol reagent using the manufacturer’s in-

(gD1168; Figure 2). Sequence analysis shows that thestructions. Following the isopropanol precipitation the RNAtruncated transcript results from the skipping of allpellet was resuspended in 100 ml reverse transcriptase buffer

with 100 mm dNTPs, 40 units RNAsin (Promega, Madison, other alternatives and the direct splicing of constitutiveWI), 200 units SuperScript II reverse transcriptase (GIBCO- exons 10 and 12 in the IFM. Using a PCR primer de-BRL, Gaithersburg, MD), and an antisense oligonucleotide signed to specifically assay for exon 10–12 skip splicing,primer to Mhc exon 12. Reverse transcription reactions were

we also find that the gD1168 transgene produces a skip-at 428 for 1 hr. For the primary PCR reaction, 5 ml of thespliced transcript only in the IFM and not in larvalreverse transcriptase reaction were used in a 100-ml reaction

under the following conditions: 1 cycle at 958 for 5 min; 30 muscles where exon 11e is not normally utilized. Thus,cycles of 958 for 30 sec, 558 for 1 min, 728 for 1 min; and 1 in the absence of sequence information required tocycle of 748 for 4 min. Five microliters of the primary PCR make the correct alternative exon selection in the IFM,reaction was used for the secondary PCR reaction and the

the splicing machinery defaults to an exon 10–12 skip-same PCR cycle profile was followed. RNAs from larval musclessplicing pathway, further supporting our data that exonwere prepared identically, except that 10–15 first instar larvae

of each genotype were directly homogenized in RNAzol prior 11e splicing is controlled by IFM-specific splicing ma-to RT-PCR analysis. The sequences for all of the oligonucleo- chinery.tide primers used in these experiments are presented below: Distal intronic sequences have IFM-specific and

global regulatory activities in exon 11e splicing: ExonExon 2 sense: GACTCGAAGAAGTCTTGCTGExon 12 inner antisense: CTGACCCAGGACACCGGCGCG 10–12 skip splicing provides a sensitive method for mon-Exon 12 outer antisense: GGACATGATCTTGCCCAGACGC itoring disruptions in IFM-specific splicing and we haveSkip-specific primer: CTACGCCACAAGAAGGCG applied this assay in combination with a series of mu-

tated minigene constructs to identify further cis-actingelements required for the IFM-specific splicing of exon

RESULTS11e. A large conserved intronic domain in the exon 11domain (CIE3; Figure 1) located between exon 11d andDisruption of exon 11e splicing leads to exon 10–12

skip splicing in the IFM: Previously, we established that exon 12 was examined earlier for its role in directingIFM-specific splicing of exon 11e (ICR; Standiford eta Mhc minigene containing the entire exon 11 domain

(gD1048; Figure 1B) has sufficient regulatory sequences al. 1997), and deletion of this element did not altersplice choice specification of any other alternative exonto direct the correct alternative slicing of exon 11 alter-

natives in their appropriate muscles (Standiford et al. when deleted. However, b-galactosidase expression,which is a function of the normal processing of the1997). Specifically, RT-PCR and LacZ reporter gene

analysis were used to show that the selection of alterna- transgene, was found to be suppressed in the absenceof CIE3, indicating a loss of processing efficiency. Antive exons from transcripts arising from the gD1148

minigene exactly matched the use of exons from the examination of this effect here with the use of the skip-splicing assay shows that the observed loss of splicingendogenous Mhc gene transcripts. For instance, exon

11e from the gD1048 minigene was used exclusively in efficiency in a CIE3 (gD1120) deletion background ac-tually results from the activation of skip splicing andthe IFM and the use of no other exon was detectable

262 D. M. Standiford et al.

Fig

ure

1.—

(A)

Dia

gram

ofth

eD

roso

phila

36B

Mhc

gen

e,sh

owin

gth

epo

siti

onof

the

13co

mm

on,

5al

tern

ativ

e,an

dsi

ngl

edi

ffer

enti

ally

splic

edex

ons.

Als

osh

own

are

the

MH

Cdo

mai

ns

that

are

enco

ded

byth

eex

ons,

incl

udin

gth

em

yosi

nm

otor

dom

ain

,th

elig

ht

chai

nbi

ndi

ng

regi

on,

and

the

MH

Cro

ddo

mai

n.T

he

myo

sin

rod

prot

ein

(Mrp

)ge

ne

isin

tegr

alto

Mhc

and

begi

ns

inM

hcin

tron

12.

(B)

Dia

gram

sof

the

pare

nta

lM

hcm

inig

ene

con

stru

ctus

edin

this

stud

y.T

he

gD10

48co

nta

ins

the

enti

reex

on11

dom

ain

,w

ith

five

alte

rnat

ives

that

are

flan

ked

byco

mm

onex

ons

10an

d12

.M

hc

exon

s1

and

2,w

hic

hen

com

pass

the

Mhc

prom

oter

dom

ain

,ar

efu

sed

toth

e59

end

ofex

on10

and

the

intr

on12

and

apo

rtio

nof

exon

13fu

sed

toth

eL

acZ

repo

rter

are

incl

uded

atth

e39

end

ofth

eco

nst

ruct

.Spl

icin

gpr

oduc

tsar

eas

saye

dw

ith

RT

-PC

Rus

ing

prim

erse

tsth

atsp

ecif

ical

lyam

plif

yth

em

inig

ene

tran

scri

pt(E

2/E

12)

acro

ssth

eex

on11

dom

ain

toid

enti

fyco

rrec

tly

splic

edpr

oduc

tsba

sed

onsi

ze.

Th

eex

on11

inte

rval

con

tain

sC

IEs

and

thos

est

udie

dh

ere

are

show

nin

the

gD10

48tr

ansg

ene.

CIE

1an

dC

IE2

are

39of

the

exon

11do

nor

inth

eex

on11

ein

tron

,wh

ileth

ela

rge

CIE

3el

emen

tis

dist

ally

posi

tion

ed20

0n

t59

ofth

eex

on12

acce

ptor

.(C

)L

ist

ofco

nse

rved

intr

onic

elem

ents

impo

rtan

tfo

rex

on11

esp

licin

gin

the

IFM

.C

IE1

isid

enti

cal

betw

een

D.

mel

anog

aste

ran

dD

.vi

rilis

and

con

sist

sof

are

peat

edA

TG

TA

CC

elem

ent.

Th

eC

IE2

elem

ent

isa

sin

gle

elem

ent

loca

ted

wit

hin

24n

tof

CIE

1in

D.

viri

lisan

dD

.m

elan

ogas

ter.

CIE

3is

ala

rger

elem

ent

wit

hse

vera

ldo

mai

ns

ofh

igh

con

serv

atio

n(s

had

ed).

CIE

3al

soco

nta

ins

thre

eA

TC

Cre

peat

san

dan

A/T

-ric

hdo

mai

n.

263Muscle-Specific Alternative Splicing in Drosophila

CIE3 splicing enhancer and its loss leads to the activa-tion of skip splicing in the IFM. However, alternativespliced exons are often regulated through the use ofESEs (Liu et al. 1998; Nagel et al. 1998; Zheng et al.1998; Gersappe and Pintel 1999). The possible func-tion of such regulatory elements in exon 11 alternativesplicing has been examined previously, but only withrespect to splice choice specificity. These earlier resultsshowed that swapping alternative exons also convertedtheir use (Standiford et al. 1997), indicating that exonposition and not its specific sequence determined itsmuscle-specific selection. Further, although all alterna-tive exon donor sites are nonconsensus, but well con-served and unique to each alternative, they were foundto not influence exon selection. However, while theseexonic elements are not providing specificity informa-tion, it is possible that they are important as splicingenhancers, similar to the CIE3 element. To test thisFigure 2.—Exon skipping occurs in the absence of IFM-

specific exon 11e sequences. When the IFM-specific exon 11e possibility, we used the exon 10–12 skip-splicing assayalternative is deleted in gD1168, RT-PCR using the exon 2–12 to examine the effects of exon and donor swaps on IFM-primer set detects only a truncated transcript in the IFM, specific splicing. As shown in Figure 4, we found that,although this analysis shows the expected size of the transcript

in the context of the complete exon 11 domain, anin the IFM of flies expressing the wild-type gD1048 transgene.exon swap involving replacement of exon 11e with 11bThe size of this transcript is consistent with a skip-spliced

product, which was confirmed by sequencing (skip product). (gD1222) does not promote exon 10–12 skip splicingA PCR primer (skip primer) that specifically amplifies skipped in the IFM, although 11b is now selected for inclusionproducts was able to detect the skip product in the IFM of in the IFM (Standiford et al. 1997). The replacementthe gD1168 flies, but not in larval muscles or in the IFM of

of the highly conserved exon 11e donor with that fromflies expressing the wild-type gD1048 minigene, showing theexon 11b (gD1105) or a consensus donor (gD1090)initiation of the skip-splicing pathway in the IFM in the absence

of required cis-acting sequence information. also does not induce the skipping reaction or changethe use of exon 11e in the IFM (Standiford et al. 1997).Thus, consistent with our earlier findings, neither exon

a routing of transcripts into this pathway (Figure 3). 11e nor its unique nonconsensus splice donor are re-Further, the loss of CIE3 disrupts splicing only in the quired for the efficient selection and splicing of thisIFM, and transcripts arising from gD1120 in the TDT, exon in the IFM. Therefore, the sequence informationwhich utilizes exon 11b, are normally spliced and no required to direct IFM-specific exon 11e splicing mustskip product is detected. Because our previous results reside in the exon 11 intronic sequence. In addition toshowed that no other alternative is aberrantly included CIE3, we identified two other conserved elements withinin the IFM in the absence of CIE3 (Standiford et al. this intronic domain, CIE1 and CIE2 (Standiford et1997), these data indicate that CIE3 is required to spe- al. 1997), and these sequences have been subjected tocially enhance exon 11e splicing in the IFM. further mutagenesis experiments to identify their role

Interestingly, while the loss of CIE3 appears to disrupt as potential splicing regulatory elements.the splicing of only exon 11e, a larger 39 deletion that CIE1 and CIE2 elements have distinct negative andremoves 11c through CIE3 was found to have more positive regulatory functions for IFM-specific, alterna-global effects on exon 11 alternative splicing (gD1242; tive splicing of exon 11e: The CIE1 element is proximalFigure 3). This deletion disables the normal processing to exon 11e and consists of a twice-repeated ATGTACCof all remaining exons (11e, 11a, 11b) and only skip- sequence in D. melanogaster and D. virilis (Figure 1C), butspliced products are detected in the IFM and larval is represented as a single element in D. hydei (Miedema etmuscles. In contrast, a further deletion (gD1060) that al. 1994). CIE2 is a complex element with the consensusleaves exon 11e as the only alternative is normally AGTGCTGTG/CT, which is separated from CIE1 by z20spliced in the IFM, but is skipped in all other muscles nt in D. melanogaster and D. virilis, but is contiguous with(see Figure 6). Thus, sequences deleted by the gD1242 CIE1 in D. hydei. To determine whether CIE1 and CIE2construct appear to be important in coordinating the elements are required for exon 11e splicing in the IFM,selection of alternatives in a multi-exon context. We mutations were introduced into CIE1 and CIE2 in therefer to this activity as the splicing coordinator (SC). gD1048 background, and transcripts from IFM of trans-

Exon 11e splice selection is controlled exclusively genic flies were tested for exon 11e splicing and skipthrough intronic elements: As seen in Figure 3, exon splicing using RT-PCR. As shown in Figure 5A, the sub-

stitution of a random linker in CIE1 (CCGTCCTTC11e selection in the IFM is specifically promoted by the

264 D. M. Standiford et al.

Fig

ure

3.—

Sequ

ence

sdi

stal

toex

on11

eh

ave

IFM

spec

ific

and

glob

alre

gula

tory

acti

viti

es.A

lth

ough

the

com

plet

eM

hc

exon

11in

terv

alfa

ith

fully

dire

cts

exon

11e

splic

ing

inth

eIF

Min

the

gD10

48tr

ansg

ene

and

no

skip

prod

ucts

are

dete

cted

,a

dele

tion

ofth

edi

stal

con

serv

edin

tron

icel

emen

t,C

IE3,

ingD

1120

indu

ces

exon

skip

pin

g.T

his

effe

ctis

spec

ific

for

the

IFM

,an

dR

NA

sco

llect

edfr

omth

eT

DT

that

are

assa

yed

byR

T-P

CR

don

otco

nta

inan

ysk

ippr

oduc

t.T

he

gD12

42tr

ansg

ene

isun

able

todi

rect

nor

mal

splic

ing

ofal

tern

ativ

eex

ons

inal

lm

uscl

es,

incl

udin

gth

eIF

Man

dla

rval

mus

cles

.N

orm

alsp

licin

gis

rest

ored

inth

eIF

Min

the

furt

her

dele

ted

gD10

60co

nst

ruct

,al

thou

ghex

onsk

ippi

ng

occu

rsin

all

oth

erm

uscl

esin

clud

ing

larv

alm

uscl

es(s

eeFi

gure

5).

Th

ere

lati

vele

vel

ofsk

ipsp

licin

gin

duce

dby

each

con

stru

ctis

pres

ente

dat

the

righ

tof

each

con

stru

ctdi

agra

mfo

rth

isan

dsu

bseq

uen

tex

peri

men

ts,w

her

e(-

--)

equa

lsn

osk

ipsp

licin

gis

dete

cted

,wh

ile(1

11

)in

dica

tes

stro

ng

indu

ctio

nof

skip

splic

ing.

265Muscle-Specific Alternative Splicing in Drosophila

Fig

ure

4.—

Spec

ific

exon

san

dn

onco

nse

nsu

ssp

lice

don

ors

are

not

requ

ired

for

IFM

exon

11e

splic

ing.

To

exam

ine

the

role

ofco

nse

rved

splic

edo

nor

san

dex

onic

sequ

ence

s,tr

ansc

ript

sar

isin

gin

the

IFM

from

con

stru

cts

that

con

tain

edan

exon

11e

toex

on11

bsw

ap(g

D12

22),

anex

on11

eto

exon

11b

splic

edo

nor

con

vers

ion

(gD

1105

),or

anex

on11

esp

lice

don

orco

nve

rsio

nto

con

sen

sus

(gD

1090

)w

ere

anal

yzed

wit

hR

T-P

CR

and

the

skip

spec

ific

prim

ers.

Inea

chca

se,h

owev

er,o

nly

the

nor

mal

lypr

oces

sed

tran

scri

ptw

asde

tect

ed.

266 D. M. Standiford et al.

Figure 5.—(A) IFM-specific regulatory elements have context-dependent activities. When CIE1 or CIE2 substitution mutationswere introduced into the gD1048 transgene (gD1175 and gD1176, respectively), there was no effect on exon 11e use in the IFM,as measured by the detection of the exon 11 spliced transcripts and the absence of exon skipping. The combination of the CIE2and CIE3 mutations (gD1320) did not enhance skip splicing over that seen in the CIE3 deletion alone (gD1120; Figure 3). (B)CIE1 and CIE2 have IFM-specific regulatory functions. The truncated Mhc minigene, gD1060, faithfully directs the inclusion ofexon 11e in the IFM and contains CIE1 and CIE2. The replacement of CIE2 with random sequence in gD1254 results in a stronginduction of skip splicing as measured both by the exon 2–12 and skip-specific primers. In contrast, the substitution of CIE1 inthe gD1256 did not effect normal splicing or induce skip splicing. The combination of CIE1 and CIE2 mutations in the gD1265transgene resulted in the return of normal splicing to near wild-type levels. (C) The single or combined loss of CIE1 or CIE2does not lead to the activation of 11e splicing in larval muscles, indicating that these elements function specifically in the IFM.

267Muscle-Specific Alternative Splicing in Drosophila

TCCAC; gD1175) or CIE2 (GAACTATCCC; gD1176)does not promote exon 11e skip splicing or lead to themisexpression of any other alternative in the IFM (notshown), suggesting that these CIE elements do not par-ticipate in splicing regulation in this context or areredundant with other elements in the gD1048 trans-gene.

However, when further examined using the truncatedgD1060 background, both CIE1 and CIE2 were foundto be critical for the proper use of exon 11e in the IFM.When the same CIE2 mutation from gD1176 was placedin the truncated context (gD1254; Figure 5B), therewas a significant loss of exon 11e splicing in the IFM,with z50% of the transcripts undergoing exon 10–12skip splicing, while the substitution of CIE1 with randomsequence did not alter the IFM-specific splicing of exon11e or induce any detectable skip splicing (gD1256;Figure 5B). However, when the CIE1 and CIE2 muta-tions were combined in the gD1265 transgene, skip Figure 6.—CIE3 is a IFM-specific splicing enhancer forsplicing was greatly reduced from that observed in the exon 11e. Transcripts arising from the gD1152 construct areCIE2 substitution alone. These results indicate that CIE2 properly regulated for the specific inclusion of exon 11e in

the IFM and no skipping is detected. The removal of CIE2is required to promote exon 11e use in the IFM and thatwhen CIE3 is intact in the gD1357 transgene does not alterCIE1 can act to repress this activity. The substitutions ofthe normal use of exon 11e in the IFM, nor does the concur-

CIE1 and CIE2 made here do not result in the activation rent loss of CIE1 and CIE2 in the gD1356 construct, demon-of splicing in larval muscles (Figure 5C) or in the TDT strating that the CIE3 element can act as an IFM-specific splic-(not shown), showing that these substitutions do not ing enhancer for exon 11e. Further, when only the 59 half of

CIE3 is included in the gD1362 construct, some skipping oc-themselves confer gain-of-function effects, and se-curs but at a level below that found in gD1254 (Figure 5),quence analysis of the spliced products revealed prod-where CIE2 and CIE3 are removed, suggesting that the CIE3

ucts that resulted from only exon 11e to 12 (normal) splice-enhancing activity is localized to the 59 domain of theor exon 10 to 12 (skip) splice reactions, suggesting that element.no new splice sites were added or activated by the CIE1and CIE2 substitutions. Thus, these mutations appear

of the CIE2 splicing enhancer (gD1356), exon 11e isto specifically affect the activity of these elements.normally spliced in the IFM and no exon 10–12 skippingWhile CIE2 appears required for the normal utiliza-is detected, indicating that CIE3 can functionally substi-tion of exon 11e, particularly in the presence of CIE1,tute for CIE2 in this context. Consistent with this obser-these experiments also show that normal splicing canvation is the fact that CIE3 also directs the inclusion ofoccur in its absence, suggesting either that the alterna-exon 11e into the IFM in the absence of both CIE1 andtive splicing regulatory mechanism also includes addi-CIE2 (gD1357). Further, when the 39 half of CIE3 istional positive-acting regulatory elements found else-deleted in combination with the CIE2 substitutionwhere in the gD1060 interval or that a default splicing(gD1362), some skipping occurs, but this appears lessmechanism that includes any alternative exon is en-than that seen in the gD1254 construct (Figure 5),gaged in the absence of CIE1 and CIE2. This secondwhere CIE3 is fully deleted. Thus, CIE3 appears to en-possibility, however, is not favored since a construct thathance the ability of the splicing apparatus to recognizeis similar to gD1060 except that it contains exon 11bexon 11e in the IFM in the absence of CIE1 and CIE2and its flanking introns is completely skipped in theand independently of any additional information con-IFM (not shown).tained in the interval removed by the gD1060 deletion.CIE3 is a positive regulator of IFM-specific exon 11eInterestingly, the 59 domain of the CIE3 is well con-splicing: CIE3 is required in the context of the full-served and contains several purine-rich elements thatlength exon 11 to properly promote the inclusion ofcould serve as sites for interactions with SR proteinsexon 11e in the IFM. The ability of this element to(Schaal and Maniatis 1999).function in a truncated context was determined in Fig-

ure 6 to test whether CIE3 acts directly with exon 11eor whether additional elements within the domain are DISCUSSIONalso needed for its function. When placed into a gD1060

In this study we have utilized transgenic analysis andbackground where both CIE1 and CIE2 are intactmutagenesis to identify the intronic regulatory elements(gD1152), no misexpression or skipping of exon 11ein Mhc transcripts that control the muscle-specific splic-can be detected. When CIE3 is present in the absence

268 D. M. Standiford et al.

ing of the alternative exon 11, which encodes the “con- IFM specificity of exon 11e splicing could involve addi-tional negative regulatory factors that repress the splic-verter domain” of Drosophila MHC isoforms (Bern-

stein and Milligan 1997). Experiments have been ing of other exon 11s in the IFM and/or repress exon11e splicing in non-IFM muscles. At this time we do notdirected specifically to the analysis of regulatory ele-

ments that control the splice choice specificity of exon favor this possibility since, to date, we have not identifiedmutations in regulatory elements that induce the misex-11e, which is spliced exclusively in the highly specialized

IFM (Hastings and Emerson 1991). These regulatory pression of exon 11e in non-IFM muscles or misexpres-sion of other exon 11s in the IFM. An exception to thiselements have been identified using RT-PCR assays that

detect the spliced products of transcripts arising from conclusion is the finding that the conversion of thenonconsensus exon 11e splice donor to a consensuswild-type and mutated transgenic Mhc exon 11 mini-

genes in the IFM. The results of our analyses reveal that, donor leads to the exclusive selection of exon 11e inall muscles. This finding demonstrates the intrinsicgenerally, the IFM-specific use of exon 11e depends on

both positive and negative interactions that appear to weakness of the normal nonconsensus 59 splice donorsequences and suggests that the activation of the donoract either locally on exon 11e or globally across the

entire exon 11 domain. Specifically, we have identified is the regulatory focus of the alternative splicing mecha-nism. Thus, our model of exon 11 alternative splicingfour exon 11 splicing regulatory elements that have

distinct functions. Two elements, CIE1 and CIE2, are regulation is predicated upon the muscle-specific activa-tion of donor sites to effect the splicing of the appro-located immediately 39 of exon 11e and act locally on

IFM-specific exon 11e splicing in the absence of the priate alternative exon.Conserved intronic elements are specific regulatorsother four alternative exon 11s. Independent of CIE1

and CIE2 are two distal elements, CIE3, which also posi- of exon 11e: The ability of CIE1 to repress the selectionof exon 11e occurs only in the IFM, and the removaltively directs the use of exon 11e in the IFM, and a SC,

which appears necessary to facilitate alternative splicing of this element does not lead to the activation of exon11e splicing in other muscles even in situations whereof exon 11e when in the context of multiple alternative

exon 11s. Therefore, a hierarchy of regulatory mecha- exon 11e is the only alternative (gD1256, Figure 5).Typically, splicing repressors function to prevent thenisms controls the IFM-specific splicing of exon 11e.

These data extend our previous findings to provide com- use of alternative exons in inappropriate tissue, andwhat role the IFM-specific repression of exon 11e splic-pelling evidence that the IFM splicing machinery is ex-

quisitely specific for splicing of exon 11e in the IFM. ing plays is an intriguing question. Given the lengthof the exon 11 domain and number of alternatives itExon 11e splicing involves a IFM-specific mechanism:

In contrast to other known alternatively spliced tran- contains, it is possible that this component of the mecha-nism is required to regulate and coordinate the timingscripts, where competitive interactions involving splice

site strengths or the abundance and state of various of exon 11e splicing during the period when other alter-natives in the domain are being transcribed, whichbinding proteins can influence the balance of exon

selection within a specific tissue, alternative splicing of serves to prevent the inappropriate definition of theexon 11e donor until the strong exon 12 splice acceptorMhc exon 11e appears to involve discrete, muscle-spe-

cific interactions that ensure the exact and exclusive becomes available (Robberson et al. 1990). Interest-ingly, mutual exclusivity is stringently maintained amonguse of the correct exon. This property is clearly shown

in the splicing behavior of the gD1168 transgene, where the exon 11 alternatives, and none of the mutationsthat we have examined lead to the splicing of multipleexon 11e alone is removed (Figure 2). In this case, the

IFM splicing machinery does not select any other exon alternative exons. The ability to repress exon definitionuntil the entire interval is transcribed is potentially part11 in the transgene transcript but instead completely

defaults to the skip-splicing mode, showing that the IFM of this mechanism and could be a feature of all exon11 alternatives.has an intrinsic requirement for a specific alternative

exon and cannot replace this with another in its ab- CIE2 has an activity reciprocal to that of CIE1 andlikely positively regulates exon 11e splicing. This modelsence. The same also appears true for other non-IFM

muscles, which do not utilize exon 11e in the transcripts is consistent with the observation that exon 11e is notefficiently spliced in the absence of CIE2 in the gD1254from the gD1060 transgene, but instead default to skip

splicing. transgene, resulting in exon 10–12 skip splicing in theIFM. We find it particularly significant that the CIE1Exon 11e is positively selected in the IFM: The basis

for the IFM exclusivity of exon 11e splicing likely and CIE2 sites are positioned immediately adjacent toone another in D. hydei and are only 24 nt apart in D.involves exon 11e-specific splicing factors that are

uniquely present in the IFM and absent in all other melanogaster and D. virilis, consistent with the possibilityof interactions between these elements. However, themuscles. We hypothesize that such IFM-specific factors

would interact with the CIE elements and potentially fact that IFM-specific use of 11e still occurs in the ab-sence of CIE1 and CIE2 indicates that these elementsother intronic regulatory sequences in the exon 11 do-

main to promote exon 11e splicing. Alternatively, the do not encompass all the information for 11e activation,

269Muscle-Specific Alternative Splicing in Drosophila

although our current data show that these will also re-side in the intronic sequence found in gD1060. Further,we have not directly tested the role of secondary struc-ture in exon 11e splicing and do not exclude the possi-bility that it plays a role in CIE1 and CIE2 function inparticular or in exon 11 alternative splicing generally.However, stable RNA structures are not predicted toform within or between CIE1 and CIE2, suggesting thatthese elements do not themselves play a direct rolein aspects of exon 11e splicing that may be regulated Figure 7.—CIE-mediated regulation of Mhc exon 11e inthrough secondary structure. the IFM. A model for the role of intronic elements in the

CIE3 is a large element located at the 39 end of the regulation of exon 11e alternative splicing in the IFM is basedon the selective enhancement in the strength of the exonexon 11 domain that is required to direct the efficient11e nonconsensus donors (see text). CIE2 acts as a positiveuse of exon 11e when the entire intron is present. Ourregulator of this event and is predicted to bind factors thatanalysis of the truncated exon 11 background demon- will either directly or indirectly attract the U1 snRNP to the

strates that this element also acts as a splicing enhancer 11e donor. The CIE1 element has an IFM-specific repressorto direct exon 11e use in the IFM and can substitute activity perhaps required to coordinate the timing of exon

11e donor utilization relative to other splicing processes. CIE1for CIE2 in this context. However, while both CIE2and CIE2 are positioned close to each other, and factors thatand CIE3 appear to act as exon 11e-specific splicingrecognize CIE2 might inactivate CIE1 repression. CIE3 is re-enhancers, their dissimilar composition and positions quired for high-fidelity use of exon 11e in the presence of

within the primary transcript suggest that these two ele- other exon 11s and can substitute for CIE2 in the truncatedments function through separate mechanisms. For in- “gD1060” background. This element might directly affect the

strength of the exon 11e splice donor, but its proximal positionstance, while CIE2 appears to be positioned where itrelative to the presumptive exon 12 branch point sequencecan interact with the splice donor of exon 11e, the(BPS) suggests that the activity of CIE3 promotes interactionsproximity of CIE3 to the putative branchpoint (AUCU between the 11e donor and the exon 12 acceptor. Deleting

AAC) and polypyrimidine tract for exon 12 might indi- the exon 11c–CIE3 interval results in complete loss of muscle-cate that it acts to influence the activity of these elements specific alternative exon 11 splicing, as evidenced by skip splic-

ing in all muscles. Thus, this domain appears to contain an SCin the IFM.activity that is required to promote the selection of individualAs seen in Figure 3, the loss of the domain containingexons and to suppress skip splicing when multiple alternativeexon 11c through CIE3 (gD1242) results in the failure exon 11s are present.

of either remaining alternative (11e and 11b) to beprocessed and the induction of skip splicing in all mus-cles. Since a larger deletion that also removes exon 11b suppress exon skipping between constitutive exons or

kinetic processes that favor formation of spliceosomes(gD1060) is permissive for exon 11e splicing in theIFM, these findings suggest that information removed on alternative exons over formation of spliceosomes for

flanking constitutive exons. The results of our mutagen-in gD1242 is important for splicing regulation whenmultiple alternatives are present. How such a SC might esis studies of IFM-specific exon 11e splicing regulation

have identified splicing regulatory elements that appearfunction is unclear, but it may act to repress competitionor other interactions among multiple alternatives that to fall into both the fidelity and differential selection

classes of regulatory processes. A model to account forprevent efficient recognition of any alternative, which,in turn, promotes exon skipping. Experiments are in the role of the elements described here is presented in

Figure 7.progress to localize the SC sequence element to defineits function in Mhc exon 11 alternative splicing. Multiple strategies exist to direct Mhc alternative

splicing: The cis-mechanism that directs the alternativeIt is clear that a complex and interconnected regula-tory hierarchy is required to ensure the proper selection splicing for several different Mhc alternative exons has

been examined (Hodges and Bernstein 1992; Standi-of exon 11e and that perturbations in this regulationlead to alternative exon skipping. With this consider- ford et al. 1997; Davis et al. 1998; Hodges et al. 1999),

and from these studies it has become clear that a varietyation in mind, alternative exon skipping could reflectdisruptions in two classes of splicing regulatory proc- of mechanisms exist to regulate the alternative splicing

of exons from different alternative groups. For instance,esses: (1) fidelity mechanisms that control the specificityof alternative exon splicing (e.g., interaction of muscle- our work here indicates that exon 11 selection is medi-

ated through 59 donor enhancement, while the selec-specific splicing factors, activators, and repressors withalternative exon-specific regulatory sequences) and (2) tion of exon 7 alternatives appears to proceed through

a mechanism that is directed at strengthening the 39alternative/constitutive exon selection mechanisms thatcontrol the differential splicing of alternative exons over acceptor (Davis et al. 1998), and the inclusion of exon

18 appears to require a specific conserved polypyrimi-the splicing between flanking constitutive exons, whichcould involve active processes such as repressors that dine tract to enhance the recognition of weak donor

270 D. M. Standiford et al.

have antagonistic effects on intronic enhancer-dependent splic-and acceptor sites (Hodges et al. 1999). The apparenting of the beta-tropomyosin alternative exon 6A. EMBO J. 16:

diversity of alternative splicing mechanisms may under- 1772–1784.George, E. L., M. B. Ober and C. P., Emerson, Jr., 1989 Functionallie the observation that there appear to be no require-

domains of the Drosophila melanogaster muscle myosin heavy-ments for the coexpression of alternatives from differentchain gene are encoded by alternatively spliced exons. Mol. Cell.

exon groups (Hastings and Emerson 1991) and may Biol. 9: 2957–2974.Gersappe, A., and D. J. Pintel, 1999 CA- and purine-rich elementsbe important in conferring a greater flexibility to gener-

form a novel bipartite exon enhancer which governs inclusionate MHC isoform diversity.of the minute virus of mice NS2-specific exon in both singly and

A surprising result from our study is the stringent doubly spliced mRNAs. Mol. Cell. Biol. 19: 364–375.Gooding, C., G. C. Roberts, G. Moreau, B. Nadal-Ginard andrequirement by the IFM for exon 11e and the discrete

C. W. Smith, 1994 Smooth muscle-specific switching of alpha-shift to a skip-splicing pathway when required regulatorytropomyosin mutually exclusive exon selection by specific inhibi-

elements such as CIEs are replaced or removed. The tion of the strong default exon. EMBO J. 13: 3861–3872.Gooding, C., G. C. Roberts and C. W. J. Smith, 1998 Role of ansingle response mode of exon skipping when con-

inhibitory pyrimidine element and polypyrimidine tract bindingfronted with a number of different regulatory defectsprotein in repression of a regulated a-tropomyosin exon. RNA

provides a unique opportunity to define multiple com- 4: 85–100.Hastings, G. A., and C. P., Emerson, Jr., 1991 Myosin functionalponents of the alternative splicing apparatus in the IFM

domains encoded by alternative exons are expressed in specificusing genetic screens designed to identify skip splicing.thoracic muscles of Drosophila. J. Cell Biol. 114: 263–276.

Having defined the specific intronic and splice donor Hodges, D., and S. I. Bernstein, 1992 Suboptimal 59 and 39 splicesites regulate alternative splicing of Drosophila melanogaster my-elements that are required for IFM-specific exon 11eosin heavy chain transcripts in vitro. Mech. Dev. 37: 127–140.splicing, the challenge now is to identify the splicing

Hodges, D., R. M. Cripps, M. E. O’Connor and S. I. Bernstein,factors that determine the precise muscle specificity of 1999 The role of evolutionarily conserved sequences in alterna-

tive splicing at the 39 end of Drosophila melanogaster myosinalternative exon 11 splicing in Drosophila.heavy chain RNA. Genetics 151: 263–276.

We thank Beth Bucher (University of Pennsylvania) for advice and Labourier, E., E. Allemand, S. Brand, M. Fostier, J. Tazi et al.,comments on the manuscript and members of the laboratory for their 1999 Recognition of exonic splicing enhancer sequences byinterest and discussion throughout the course of this project. This the Drosophila splicing repressor RSF1. Nucleic Acids Res. 27:

2377–2386.work was supported by a U.S. Army Breast Cancer Initiative Postdoc-Libri, D., A. Piseri and M. Y. Fiszman, 1991 Tissue-specific splicingtoral Fellowship (to D.M.S.), a Muscular Dystrophy Association Post-

in vivo of the beta-tropomyosin gene: dependence on RNA sec-doctoral Fellowship (to M.B.D.), an American Cancer Society grantondary structure. Science 252: 1842–1845.NP 841 (to C.P.E.), and a National Institutes of Health R01-AR42363

Lin, C. H., and J. G. Patton, 1995 Regulation of alternative 39 splicegrant (to C.P.E.).site selection by constitutive splicing factors. RNA 1: 234–245.

Liu, H. X., M. Zhang and A. R. Krainer, 1998 Identification offunctional exonic splicing enhancer motifs recognized by individ-ual SR proteins. Genes Dev. 12: 1998–2012.LITERATURE CITED

Lou, H., D. M. Helfman, R. F. Gagel and S. M. Berget, 1999 Polypy-rimidine tract-binding protein positively regulates inclusion ofAshburner, M., 1989 Drosophila: A Laboratory Handbook. Cold Spring

Harbor Laboratory Press, Cold Spring Harbor, NY. an alternative 39-terminal exon. Mol. Cell. Biol. 19: 78–85.Miedema, K., H. Harhangi, S. Mentzel, M. Wilbrink, A. Akhma-Bandman, E., 1992 Contractile protein isoforms in muscle develop-

ment. Dev. Biol. 154: 273–283. nova et al., 1994 Interspecific sequence comparison of the mus-cle-myosin heavy-chain genes from Drosophila hydei and Dro-Bernstein, S., and R. Milligan, 1997 Fine tuning a molecular

motor: the location of alternative domains in the Drosophila sophila melanogaster. J. Mol. Evol. 39: 357–368.Mullen, M. P., C. W. Smith, J. G. Patton and B. Nadal-Ginard,myosin head. J. Mol. Biol. 217: 1–6.

Chabot, B., 1996 Directing alternative splicing: cast and scenarios. 1991 Alpha-tropomyosin mutually exclusive exon selection:competition between branchpoint/polypyrimidine tracts deter-Trends Genet. 12: 472–478.

Chen, C. D., R. Kobayashi and D. M. Helfman, 1999 Binding of mines default exon choice. Genes Dev. 5: 642–655.Nadal-Ginard, B., C. W. Smith, J. G. Patton and R. E. Breitbart,hnRNP H to an exonic splicing silencer is involved in the regula-

tion of alternative splicing of the rat beta-tropomyosin gene. 1991 Alternative splicing is an efficient mechanism for the gen-eration of protein diversity: contractile protein genes as a modelGenes Dev. 13: 593–606.

Clouet D’Orval, B., Y. D’Aubenton-Carafa, J. M. Brody and E. system. Adv. Enzyme Regul. 31: 261–286.Nagel, R. J., A. M. Lancaster and A. M. Zahler, 1998 SpecificBrody, 1991 Determination of an RNA structure involved in

splicing inhibition of a muscle-specific exon. J. Mol. Biol. 221: binding of an exonic splicing enhancer by the pre-mRNA splicingfactor SRp55. RNA 4: 11–23.837–856.

Davis, M. B., J. Dietz, D. M. Standiford and C. P. Emerson, Jr., Patton, J. G., S. A. Mayer, P. Tempst and B. Nadal-Ginard, 1991Characterization and molecular cloning of polypyrimidine tract-1998 Transposable element insertions respecify alternative

exon splicing in three Drosophila myosin heavy chain mutants. binding protein: a component of a complex necessary for pre-mRNA splicing. Genes Dev. 5: 1237–1251.Genetics 150: 1105–1114.

Dominguez, R., Y. Freyzon, K. M. Trybus and C. Cohen, 1998 Crys- Perez, I., C. H. Lin, J. G. McAfee and J. G. Patton, 1997 Mutationof PTB binding sites causes misregulation of alternative 39 splicetal structure of a vertebrate smooth muscle myosin motor domain

and its complex with the essential light chain: visualization of site selection in vivo. RNA 3: 764–778.Ramchatesingh, J., A. M. Zahler, K. M. Neugebauer, M. B. Roth,the pre-power stroke state. Cell 94: 559–571.

Du, C., M. E. McGuffin, B. Dauwalder, L. Rabinow and W. Mattox, and T. A. Cooper, 1995 A subset of SR proteins activates splicingof the cardiac troponin T alternative exon by direct interactions1998 Protein phosphorylation plays an essential role in the regu-

lation of alternative splicing and sex determination in Drosophila. with an exonic enhancer. Mol. Cell. Biol. 15: 4898–4907.Robberson, B. L., G. J. Cote and S. M. Berget, 1990 Exon defini-Mol. Cell 2: 741–750.

Dye, B. T., M. Buvoli, S. A. Mayer, C. H. Lin and J. G. Patton, tion may facilitate splice site selection in RNAs with multipleexons. Mol. Cell. Biol. 10: 84–94.1998 Enhancer elements activate the weak 39 splice site of alpha-

tropomyosin exon 2. RNA 4: 1523–1536. Ryan, K. J., and T. A. Cooper, 1996 Muscle-specific splicing en-hancers regulate inclusion of the cardiac troponin T alternativeGallego, M. E., R. Gattoni, J. Stevenin, J. Marie and A. Expert-

Bezancon, 1997 The SR splicing factors ASF/SF2 and SC35 exon in embryonic skeletal muscle. Mol. Cell. Biol. 16: 4014–4023.

271Muscle-Specific Alternative Splicing in Drosophila

Schaal, T. D., and T. Maniatis, 1999 Selection and characteriza- constitutive exons can influence alternative splicing. J. Biol.Chem. 274: 29838–29842.tion of pre-mRNA splicing enhancers: identification of novel SR

protein-specific enhancer sequences. Mol. Cell. Biol. 19: 1705– Xiao, S. H., and J. L. Manley, 1998 Phosphorylation-dephosphoryla-1719. tion differentially affects activities of splicing factor ASF/SF2.

Southby, J., C. Gooding and C. W. Smith, 1999 Polypyrimidine EMBO J. 17: 6359–6367.tract binding protein functions as a repressor to regulate alterna- Xu, R., J. Teng and T. A. Cooper, 1993 The cardiac troponin Ttive splicing of alpha-actinin mutually exclusive exons. Mol. Cell. alternative exon contains a novel purine-rich positive splicingBiol. 19: 2699–2711. element. Mol. Cell. Biol. 13: 3660–3674.

Standiford, D., M. Davis, W. Sun and J. C. Emerson, 1997 Splice- Zheng, Z. M., M. Huynen and C. C. Baker, 1998 A pyrimidine-richjunction elements and intronic sequences regulate alternative exonic splicing suppressor binds multiple RNA splicing factorssplicing of the Drosophila myosin heavy chain gene transcript. and inhibits spliceosome assembly. Proc. Natl. Acad. Sci. USAGenetics 147: 725–741. 95: 14088–14093.

Stark, J. M., T. A. Cooper and M. B. Roth, 1999 The relativestrengths of SR protein-mediated associations of alternative and Communicating editor: A. J. Lopez