Alternative Splicing of mRNA as a Mode of Endocrine Regulation

B:1996. MoLILII:lI ionut high lfjllage-;ictill:~[ccl c:ilci~]llqcllzillnels b\s~JInalosta[io inc~cL1(el\is(.)l:iteci l-alarmj$cfaJNc~lr;sci 16:6000 -60 11. oidnctlmns.

Viana

F, Hillc

White RE, %honbrunn A, Atrnstrong DL: I991. Soma(ostatin stimulates Ca2 -ac[iw(cd K ‘ channels lhrough pru[ein clephus pho]-yla[ioll. Nature 35 I :570-573. Wilkinson

CF, Fcniuk

W,

19Y7. Ch~~r:tcteriz:iti(]tl nant sonlat(m(atin

PPA:

}ILInIphI-Cy

of human

reconlbi-

sst5 t-ccc ptors mediating

~i falnll,,

of’phosphuinrxi tide metabolism Br J Pha[-mace] 121:91–96.

W\att MA, Jalxie E, Fcniuk W, Humphrey PPA: 1996. Somatostatin Sstz reccplol-tmediatd inbibitiun of parietal cell I’unctic)u in rat isolated gas[ric- mucmsa. Br J Pharmxo] 1 I9:905–9 I(). Yamada Y, Post SR, Wang K, et a].: 1992a. Cloning and l“unctional cllzil-z~ctcriz~ititln0{

:, j ICIrrroLlse somat(~s(atin

exprcssc(l II hrain, gastl.oi ntesti nal tr-ao, and kidt I, ~. Proc Nzrtl Acad Sci USA 89:?5 1–255.

rcccptrr)s

), Rcisine 1 i.L1\V SF, et al.: 1992b. Sorna(wtatin recelj IIS, an expanding Rcne family cloning art [’functional character.iz,ation t,f human S5 [’R3, a prolcin couplccl to adeI]vlatc cyLI, SC. Mcrl f3ndocrinol 6:2 136-.? 142.

Yamada

Zeggari

acti\a(ion

“f tlumzin

h’i, Esteve J 1‘ R:ILIly 1, e[ al.: 1994.

Cupurif’cation 0[”: phatase \vithacti\:, tors [’II,tm r-a[ p:, branes. E+irxl]cm J Zheuy H, Bailey A, .1 Srrmato. fatin rece[l mice aI c refractO negativ, feedback ~ Endocr Im)l 11: 170

>lotein lyrosine phosd somatostatin rccclI.reatic acinal. men)3:441-448. (IIg M-H, et al.: 1997. ~rsubtype 2 knock-out to growth hormone :U.CLIZite nLW1-Oll S. MO] 1717. TEM

Wimlandet 1994). A family of snRNAs interac[ by base pairing with sequence information defining the 5‘ and 3‘ splice sites of an cxon. The consensus scquenccs for these splice sites are loose. Tbere are many examples of constitw ti\ely LIscd splice sites that deviate considerably from the consensus, whereas sequences that match the consensus may be present on a pre-mRNA and yet never used lot-splicing. When alternative splicing occLIrs, the splicing apparatus must either use a splice site not genetally recognized or ignore a splice site that is usually used. Thus, the regulation of altelnativc splicing is often discussed in tetms of splice site selec[ion. The terms “s( rong” and “weak” splice sites are often applied to sites that match or deviate from the conscrrsus, respectively. In alternative splicing, different conlbinations of exons can be derived Irolm a pre-mRNA (for examples, see Figures 3, 4, and 5). There are numerous genes \\,ith~1] (elnzltive splicing patterns, and

AlternativeSplicing of mRNA as a Mode of Endocrine Regulation Shern L. Chew

Alternative splicing ofmessenger RNA (nzRAA) is a n) ‘an.%ofregula[ing geH6’eXpt’eSSiOll and occurs in nlany genes ol”the end( ) iine system. T/liS review

covers

an

introduction

into

mRNA splicing,:.. and the meclla -

fli.sms and regulation of alterfza[ive .splicin,q.So??le xamples are di.scus.seclin which alternativelyspliced genes encode fi[,!ctionally distinct proteins. Evidence that hormones and other metal dic signals may regula[e nism.s

alternative are con.sidwed.

splicing

events

(Trends

is revie~ veal, an[~ potential

Endocrinol

Metab

mecha-

I )$)7; s:405-4

13). o

1998, Ekevier Science tnc.

Split genes were cfiscxwercxi in 1977 (Em-get et al. 1977, ChovV et al. 1977, lefi-e>s and Flavell 1977). Exons are separated bv introns in the genom ic DNA, as \vell as in the pt-imary RNA transct-ipi (prc-mRNA) (Figut-e 1). Most eukal-votic genes contain irr[rons, and [hese musl be remcwed to gi\e a mature

Shern L. Chc\\, is at the Deparlmen( of’ End(.)ct’inology, St B~\l-t}l(~](>nlc\i’s H(.rspit2Ll, LoIIdon F.C IA 7BE, United Kingdom.

7’EM Vol. 8, N(). 10, 1997

mRNA [ ]anscript i r transport from the nucleus Io the Cvt{ IIasm, where transktion inn) protein ccurs. Splicing is a very ac~ urate pbt I ]menon, with exons en if separated by precisel) joined, many’ kilobases 1“ intmn (Haukins 1988). This precisi n is achieved by premRNA sequence ii I’onmation (Figure 2) and nuclear fact( 1,>. The nuclcal--splicing app:iratus is c:i CLIthe “splicemx)mc” and is a cornplcx ~rlade up of srna}} nLl clear RhAs (snRR S) and many proteins (Moore et al. !993, Bauren and

this is an important and common mechanism f’ot- generating protein diversity and regulating gene expression in higher cukaryotes (Smith et al. 1989). The regulation ot” altcn-native splicing events by developmental and tissue-specific lac[ors is well recognized, and in some cases, the molecular mechanisms have been c[ucidated. There are severa] examalternative splicing being ples of changed by hormonal or metabolic signals (Table 1). This is a relatively new area of research, and the mechanisms are nol urtdcrstood. The general strategy used for dissecting the mechanisms of” alternative splicing is w map the sequence elements on the premRNA through which regukltion is nledialed. In higher eukalyotes, this is by constrnc[ing and transecting a “minigene” into cell lines. The minigene usually contains [be upstream and downstream constitutivc exons that flank the alternatively spliced cxons, together with all or part of the introns. The minigenc is then mutated to idcntih key sequence elements, which, in turn, may allow purification of factors interacting with those sequence elements. Some examples oi” sequence elements (other than splice site sequences) shown to enhance or inhibil alternative splice site usage are listed in Table 2. In some cases, the proleins interacting with enhancers and inhibitors have been identified.

() 1998, Elsmic] Scicnc, [nc., 1043-2 >0/98/$17.00 PII S104.3-2760(97)001 67-6

405

V

i

F, a Hnillc a B: 1996.

MOLILII:LI ion uf high

lfjllage-;ictill:~(ccl calcium channels b) s~)Inalosia[ in in acLI(el\ isolated I-al armj$cfaoid nctImns. White

RE,

I 991.

J Nc~lr;sci

%honbrunn

Soma(ostatin

16:6000–601 A, Atmstrong

stimulates

DL:

Ca2 -ac[i-

Wilkinson CF, Fcniuk W, }ILInIphI-Cy PPA: 19Y7. Ch~~r:tcteriz:iti(]t) of human recon3bisst~ t-ccc ptors mediating

acti\a(ion of’ phosphuinrxi tide metabolism Br J Pha[-mace] 121 :91–96. W>att

MA, Jal\,ie

E, Fcniuk

PPA: 1996. Somatostatin

W, Humphrey

Sstz reccplol-tme-

diated inhibition of parietal cell I’unctic)u in rat isolated gas[ric- mucx)sa. Br J Pharnxxo] 1 I 9:905–9 I (). Yamada Cloning

rcccptcr)s exprcssc(l II hrain, gastl.oi ntesti nal tr-ao, and kidt I, ~. Proc Nzrtl Acad Sci USA 89:?5 1–255.

1.

w(cd K ‘ channels lhrough pru[ein clephus pho]-yla[ioll. Nature 35 I :570-573.

nant somatos(atin”

~1falmil,, “f humzln :, j ICImouse somatos~atin

Y, Post SR, Wang K, et a].: 1992a. and l“unctional cllzil-z~ctcriz~itit)n 0{

Yamada

), Rcisine

Soma(mtatin

1

recelj

i.L1\VSF, et al.: 1992b. IIS, an expanding Rcne

family cloning art l’Llnctional character.iz,ation ()f hL[man S5 [’R3, a prolcin couplccl to adeI]vlatc cyL I, SC. Mcrl f3ndocrinol 6:2 136-.? 142. Zeggari

M, Esteve

J 1‘ R:ILIIY 1, e[ al.:

1994.

Cupurif’; cation 0[” : >lotein lyrosine phosphatase \\ith acti\:, d somatostatin rccclItors [’II[m r-a( p:I .reatic acinal. menbranes. E+irxl]cm J )3:441-448. Zheuy

H, Bailey

A, .1 LIIg M-H, et al.: 1997.

Srrmatotfat in rece[l )r subtype 2 knock-out mice aI c refractO to growth hormone negativ, feedback ~ :11.CLIZit~ nLW1-Oll S. MO] Endocr Inol 11: 170 1717. TEM

Wimlandet 1994). A family of snRNAs interac[ by base pairing with sequence information defining the 5‘ and 3‘ splice sites of an cxon. The consensus scquenccs for these splice sites are loose. Tbere are many examples of constitw Lively LIscd splice sites that deviate considerably from the consensus, whereas sequences that match the consensus may be present on a pre-mRNA and yet never used lot-splicing. When alternative splicing occLIrs, the splicing apparatus must either use a splice site not genetally recognized or ignore a splice site that is usually used. Thus, the regulation of altelnativc splicing is often discussed in tetms of splice site selec[ion. The terms “s( rong” and “weak” splice sites are often applied to sites that match or deviate from the conscrrsus, respectively. In alternative splicing, different conlbinations of exons can be derived Iro]m a pre-mRNA (for examples, see Figures 3, 4, and 5). There are numerous genes \\,ith~1] (elnzltive splicing patterns, and

Alternative Splicing of mRNA as a Mode of Endocrine Regulation Shern L. Chew

Alternative splicing of’messenger RNA (mRAA) is a n) ‘an.%ofregula[ing geH6’eXpt’eSSiOH and occurs in nlany genes ol”theend() rine system. This review covers an introduction into mRNA splicing,:.. and the meclla fli.smsand regulation of alterfza[ive .splicin,q.So??le xamples are di.scus.seclin which alternativelyspliced genes encode fi[,!ctionally distinct proteins. Evidence that hormones and other metaldic signals may regula[e alternative splicing events is revie~veal,an[~potential mecha13). o Metab \ )$)7; 8:405-4 nism.sare con.sidwed. (Trends Endocrinol 1998, Ekevier Science [nc.

Split genes were cfiscxwercxi in 1977 (Em-get et al. 1977, ChovV et al. 1977, lefi-e>s and Flavell 1977). Exons are separated bv introns in the genom ic DNA, as IVCII as in the pt-imary RNA transct-ipi (prc-mRNA) (Figut-e 1). Most eukal-votic genes contain irr[rons, and [hese musl be remcwed to give a mature

Shern L. Chc\\, is at the DeparLnlen( of’ !3ndw ct’inology, St B~\l-t}l(~](>nlc\\’sHospit2Ll, LoII don F.C I A 7BE, United Kingdom.

7’EM Vol. 8, N().

10,1997

mRNA [ ]anscript i r transport from the nucleus Io the Cvt{ IIasm, where transktion inn) protein ccurs. Splicing is a very ac~ urate p}]{ I ]menon, with exons en if separated by precisel) joined, many’ kilobases 1“ intron (Hawkins 1988). This precisi n is achieved by premRNA sequence iI I’onmation (Figure 2) and nuclear fact( II >. The nuclcal--splicing app:iratus is c:i CLIthe “splicemx)mc” and is a cornplcx ; lade up of srna}} nLlclear RhAs (snRR S) and many proteins (Moore et al. !993, Bauren and

() 1998, Elsmic] Scicnc,

this is an important and common mechanism f’ot- generating protein diversity and regulating gene expression in higher cukaryotes (Smith et al. 1989). The regulation 01”altcn-native splicing events by developmental and tissue-specific lac[ors is well recognized, and in some cases, the molecular mechanisms have been c[ucidated. There are severa] examalternative splicing being ples of changed by hormonal or metabolic signals (Table 1). This is a relatively new area of research, and the mechanisms are nol urtdcrstood. The general strategy used for dissecting the mechanisms of” alternative splicing is w map the sequence elements on the premRNA through which regukltion is nledialed. In higher eukal~otes, this is by constrnc[ing and transecting a “minigene” into cell lines. The minigene usually contains [be upstream and downstream constitutivc exons that flank the alternatively spliced cxons, together with all or part of the introns. The minigenc is then mutated to idcntih key sequence elements, which, in turn, may allow purification of factors interacting with those sequence elements. Some examples oi” sequence elements (other than splice site sequences) shown to enhance or inhibil alternative splice site usage are listed in Table 2. In some cases, the proleins interacting with enhancers and inhibitors have been identified.

[nc., 1043-2 >0/98/$1 7.00 PII S 104.3-2760(97)001 67-6

405

●

Transcription startsite

Calcitonin/Calcitonin Gene-Related Peptide

Calcitonin/Calcitonin Gene-Related Peptide (CGRP) gene splicing has been the most investigated of the alternative splicing events in the endocrine system (Figure 3). The splice from exon 3 to exon 4 of the gene occurs in the thyroid gland (and in most cell lines) and produces calcitonin. In neural tissues and F9 cells (derived from mouse teratocarcinema), however, exon 4 is skipped, and splicing occurs from exon 3 to exons 5 and 6, producing CGRP instead. This alternative splicing pattern is tightly regulated by tissue- or cell-line–specific factors. Mutational analyses have shown that regulation occurs at exon 4 (calcitonin). Even if the 3’ splice site of exon 5 (CGRP) is deleted, F9 cells are unable to splice to exon 4 (Emeson et al. 1989). This is because the 3’ splice site of exon 4 is weak. In the human gene, a U ribonucleotide (instead of an A) is used as the branchpoint (see Figure 2), whereas in the rat gene, a C is used as the branchpoint. Mutation of noncanonical rat or human branchpoint nucleotides to the consensus A results in loss of tissue- or cell-specific splicing, with all cells capable of some splicing to the calcitonin exon. In addition to the weak branchpoint of exon 4, other sequences appear to regulate the use of the calcitonin exon. Enhancer sequences within exon 4 can increase the selection of the suboptimal exon 4 branchpoint but lose their enhancing properties when the calcitonin exon 4 branchpoint is strengthened from U to A (Yeakley et al.

Genomic DNA

I RNApolymeraseII

pre-mRNA

7mG~\

mRNA

m

PolYA

/7’

Y

cap

splicing

tail

Figure1. The major wcnts of pre-mRNA processing.The pre-mRNA is transcribed from the transcriptionstartsite (mrow) on the genomic DNA template by RNA polymerase II (hatched circle). The pre-mRNA includes exons (boxes) and introns (lines). This is then Processed by the addition of’“acap (7 mG), splicing out of the introns, and addition of a poly(A) tail.

Figure2. (A) Consensus sequences of the 5’ and 3’ splice sites. The lower caselettersrepresent intronic sequence. //represents intron boundaries. Upper-case,bold letters represent exon sequences.Bold dottedlines representarbitrarylengthsof exon sequence.Dotted lines represent arbitrary lengths of inlmn sequence. The nucleotides are A, adenine; C, cytosine; G, guanine; U, uracil; N, any nucleotide; Y, pyrimidine (C or T); and R, purine (A or G). The 3’ splice site consists of three components: The undedined a represents the branchpoint, and the y(n) represents the polypyrimidine tract of 11 or more sequential pyrimidines. Recently, a new class of introns has been recognized bordered by at-ac (instead of gu-ag) with a requirement for different spliceosomal components (Tarn and Steitz 1996, Wu and Krainer 1996). (B) Splicing consists of two transeslerification reactions. Step 1 involves the breaking of the phosphate bond between the first “g” of the intron and the upstream exon (open box), by the formation of a phosphate bond between the 5’ position of the “g” and the 2’ position of the branchpoint “a.” Step 2 involves the formation of a phosphate bond between the upstream exon and downstream exon (box with central bar), with release of the intron (in the shapeof’a “lariat”). The intermediate and final reaction products can be monitored during in vitro splicing reactions with radiokibeled substrate RNAs.

1993, Van Oers et al. 1994, Zandberg et al. 1995). The downstream intron also contains sequence elements that enhance the use of the calcitonin exon (Lou et al. 1995). Factors interacting with these enhancers have been identified (Lou et al. 1996), None of these factors are tissuespecific, so their distribution alone is insufficient to explain the tissue specificity of calcitoninlCGRP alternative splicing. One possibility is that the concentration of these factors differs among tissues, resulting in differential use of the calcitonin exon. In addition to tissue-specific regulation, dexamethasone was found to change the relative levels of calcitonin/ CGRP mRNA in TT cells (medullary thyroid carcinoma derived). Control TT

@1998, Elsevier

A

3’ splice site

5’ splice site

---NNNCAG//guragu-------------------ynyuray---y(11)cag//NNN---

Substrate pre-mRNA

1 Intermediate products

1 Cnla-g Intron lariat

,

~ Final product

ScienceInc., 1043-2760/98/$17.00 PIIS1043-276O(97)OO167-6

TEM

vol.

8,

No.

10, 1997

calcitonin splice in most tissues and cells

calcitonin:CGRP mRNA ratio was an elfecl (of dexamcthasone on splicing and not on mRNA turnover (Cote and Gagel 1986).

●

CGRP splice in neural tissue and F9 cells Figure 3. A diagmm o(’ the al[crma(i\e splicing pattern of hc last fou I sons 01 ~hc czrlcitonin/ CGRP gene, Diugo}lal [iJze.s indicalc the splice choices: I“r.on]exorr 3 (o J ,r froln exon 3 to t,xons .5 aInd 6. The l>c)lv:tcienyl:~(i(>tl signals arc slIo\\m by the /e[/erA, Exor) . 4 arncf 6 ar-e considcl.ed is tc~rnlirnal cxons, as I Ihc> cnd \\,i~hpol\ (A) signals, af’tet- \\hich thc~ ~le-rm RNA transcript clczI\cd, and the PO]}(A) tail is added 1>)the enzyme pol? ( A) polyrncl SC.

cel Is bad increasing levels 0[’ calcitonin and CGRP mRNAs until 4 days in CLLturc, and thmcaf’ter both declined coordinatelv, In contrasl, lreatmcnt with dOamcthasone increased calcilonin m RNA and decreased CGRP InRNA; tb is effect

u,as r-mcrsible ii ..lcxamethasorrc ~vas withclra\vn after 4 c~OVS.Importantly, cxpminlents with acl 1~ornycin D (an inhibitor of’ tlanscripti, ~~]) showed no di lference in [he stabil ] \ of the LWJO mRNA hat the change in forms, implying

F“igure4. The insulin r-eccp(ol. pre-mRNA splicing patter-rl is shown 1, ) [he upper left, i{hel.e cxon 1I is a casse[tc won, either included 01 skippccl ([,’iagoml Ii]jt ‘I. Beneath the splicing I’iguw is a list 01”the potential func{ ional cff’eels of”alter-rati\c spl ici ! L of”exon 1 I on insulin r~ccptor func( ion. A diagram of Lhe insulin rwcptor is o) I the right, I ith the posi(ion of” the armino acid [.esicfucs encoclcd I)y ex(~n 1 1 shmn by an al-, OLI.

Insulin receptor pre-mRNA splicing pattern

Insulin Receptor

The insulin receptor gene has 22 exorrs, with alternative splicing of’ exon I 1 (Figure 4). Exon 11 is included at a low Icvcl o(” 1()[+&3@?/0 of’ insulin rcccptot- mRNAs in ]most 1issues. [n human Iivel; it is included in over 500/0 of transcripts. The inclusion of amino acid residues cm coded by cxorr 11 is of importance to the f’unclion and signaling of the insulin receptor [Figure 4; for rcf’crences, see Kc)saki et a]. ( 1995)]. In HepG2 cells (hw man hcpatoma derived), stimulation with dcxamethasonc increased the amount of cxon 11 inclusion, and this effect was reversed when dcxamethasone \vas wi[hdralvn. Exon 11 is 48 nw cleot ides (nt) in length and is bordered by an Llpstream intron of about 1.2 kb and a downstream intron of over 10 kb (Seint) et al. 1990). This small cxon size is at the lower Iimit of the range of sizes in matnmalian internal exons (Hawkins 1988) and has relatively weak splicing signals. The mechanism of skipping of cxon 11, and its increasmf inclusion with dexamethasone tr-eatment, is unknown. The small size of cxon 11, however, may cause crowding of the splicing apparatus at the splice sites and thus loss of spl ic-

u–subunits

produces isoform A Insulin binding domains Position of amino acid residues encoded by alternative exon 11

produces isoform B

Transmembrane Domain

Consequences for insulin receptor function (see right) Affinity of isoform A for insulin is 2-fold higher (i/I V;VO)

ATP binding

Isoform B couples more efficiently to signaling substrates IRS-I (in V/VO)

Regulatory region

Insulin-stimulated autophosphorylation and kinase activity are greater for isoform B (in vitro) C-terminal tail Two groups found isoform A exhibits higher internalization and recycling rate, whereas a third did not.

‘1’EMVol. 8, N(,. 10, 1997

p-subunits

C I Y98, Elswicr Scien(’. rnc., 1043-.? hO/98/$ 17.00 PIIS1043-2760(97)001 67-6

407

al

*

+

exon

A

erbActl

erbAct2

I I

I I ,,,:,,::,:

N

Figure5. Alternative splicing patterns of the thyroid hormone receptor gene (erbAal and 2) and the structure of the gene transcribed from the opposite DNA strand (Rev-ErbAa). The erbAa2 splice from the alternative 5’ splice site (’k)is indicated by diagonal lines. The area of overlap between erbAa2 and Rev-ErbAci RNAs is shown by clottedvertical lines. Open boxes, translated exons; shaded boxes, untranslated exons; horizontal lines, introns; arrows, the direction of transcription; A, polyadenylation signals. The figure is redrawn from Hastings et al. (1997). ing efficiency (“steric hindrance”). Also unknown is the pathway by which dexamethasone influences these mechanisms. Other stimuli have been shown to alter the splicing of exon 11 of the human insulin-receptor gene, including glucose and insulin (Table 1). Glucose also increases the efficiency of splicing of constitutive exons of the insulin pre-mRNA and therefore may have a more general effect on RNA splicing (Wang et al. 1997). One difficulty with this system is that it has not been excluded that inclusion of exon 11 changes the stability of the insulin receptor transcripts.

●

Protein Kinase C~

●

Another cell-line–specific example of hormonally regulated alternative splicing is in the mouse protein kinase C~ gene. Insulin stimulation switches expression from the ~1 isoform to the B2 isoform within 15 min in the mouse myocyte cell line, BC31-11 (Chalfant et al. 1995). The insulin-stimulated switch in expression does not occur in mouse adipocyte cells (3 T3/Ll ), although this cell line is insulin-responsive by other criteria (Bandyopadhyay et al. 1997). These isoforms are encoded by alternative 3’ terminal exons. Although the rapid increase in expression of transcripts containing the @2 exon is likely to be due to a change in splicing, the rapid decay of

408

exon P 1 transcripts is partly due to an increase in degradation. An analysis of the half-life of the ~1 exon (after blocking transcription with actinomycin D) showed that insulin stimulation increased the rate of decay of this RNA species. This interaction of a change in mRNA stability, with a splicing change, complicates the analysis of this system. For example, it has not yet been formally demonstrated that insulin does not dramatically increase the stability of the P2 exon transcripts (which is undetectable in unstimulated cells) while concomitantly reducing the stability of the 61 exon transcripts.

01998,

Thyroid Hormone Receptor

The last detailed example is that of thyroid hormone receptor alternative splicing. This system is important because it illustrates how alternative splicing can generate protein isoforms with opposite, and competing, functions. Also, there is some evidence that this system may be regulated by a novel mechanism, involving the transcription of an antisense RNA from the opposite strand of the genomic DNA. This lmay involve regulation by tissue differentiation factors; however, evidence for regulation by direct hormonal stimulation has not been published. Two genes (erbAa and erbA~) encode thyroid hormone receptors in verte-

I

—n

brates. Both genes have alternatively spliced mRNAs. In the case of erbAa, al or 0-2 mRNA isoforms are produced (Figure 5): The al protein product is a functional thyroid hormone receptor, whereas the a2 protein product lacks a thyroid hormone–binding domain. The ci2 protein product, however, retains the ability to bind DNA and competes for DNA binding sites with functional receptors coded by al and the P isoforms, thereby functioning as a negative regulator of thyroid hormone action (Katz and Lazar 1993). The splicing pattern of erbAa is shown in Figure 5 and involves use of an alternative 5’ splice site (shown by the asterisk) in the terminal a 1 exon. If the alternative 5’ splice site is not used, then the entire CX1exon is included in the transcript, ending in a polyadenylation signal. If the alternative 5‘ splice site is used, the transcript splices instead to the a2 exon. Transcription also occurs on the opposite strand of DNA overlapping the a2 exon, from a gene called RevErbAa. The ratio of al :a2 mRNA correlates with the levels of Rev-ErbAa RNA in different B lymphocyte cell lines, during adipocyte differentiation, and under certain culture conditions in a pituitary cell line [for references, see Hastings et al. (1997)]. It has been hypothesized that the a2 splice is controlled by Rev-ErbAa mRNA through base-pairing interactions (as these mRNAs are complementary to each other, the Rev-ErbAa RNA is effectively an endogenous antisense RNA to erbAa2 mRNA). If further evidence in favor of this hypothesis can be presented, then this will be a novel mechanism for the tight control of proteins with competing actions.

Elsevier ScienceInc., 1043-2760/98/$17.00 PIIS1043-276O(97)OO167-6

TEA4Vo[. 8, NO. 10, 1997

Table 1. Hormonal rewlation of alternative mRNA sdicinu Alternatively spliced mRNA

Stimulus

Referef~ce

Insulin receptor

Dexamethasone

Calcitonin/CGRP Protein kinasc C(3 IGF-1

Dexamethasorre Insulin GH

FGF-R TNFw PTP1 B TNFP, ~ globin Hacl hPMCA2

Cytokines 2-An~inopm-ine PDGF, EGF, bFGF

CD44

Agr-in

TPA, PDGF, IGF-I Concanavalin A [rrsulin, via HRS TGF(31, vitamin D, RA NGF

Kosah. and Webster (1993) Norgt,1 et al. (1993) NorgI ~~ I et al. (1 994a) Nor.g] ~•1 et al. (1994a) Huan, et al. ( 1996) Sell el il. (1994) Wiersl ,:1et al. (1997) Huan: et al. (1994) HLI:UI: et al. (1996) Nor-g] :, et al. (1994b) Cote :, d Gagel (1 986) Chall”: t et al. (1995) Lowe ~t al. (1 988) Che\i t al. (1 995) Zhao ~• al. (1994) Jarrol et al. (1996) Shifri and Ned (1993) Ned \ al. (1 995) Cox m .1Walter (1996) Zaclu [as and Strehler (1 996 ~ Ficht( et al. (1997) Koni: t al. (1997) Du et 1. (1997) Magr~ son et-al. (1991) Smitl .1 al. (1997)

SRp20

seTunl/cell

Junlai.

Glucose Insulin

Fibronectin EIIIB (rat) Fibronectin ED (human)

●

Src

UPR Calcium

Cvck

PhosphorylatedSplicing Factors and Spliceosome Assembly

Very 1ittle is known about the nuclea~ control of’ alter-native splice site selection during tissue difikrentiation or after hormonal stirnul i. One general approach is to irrvestigate signal ing pathways in~olwxf by specific pharmacological inhibitors, using the splicing switch as an assay. For example, the signaling pathways in~olvtxf in CD44 alternative splicing in ncuroblastorna cells have been blocked by the protein kinase C and phosphoinositol-3 kinase inhibitor-s, GF109203X, arndwortmannin (Fichtel et al. 1997), CD44 alternative splicing may also be changed by concanavalin A stimulation, by mechanisms that may involve protein kinase C and activated p21 r-as (Konig et a]. 1997). Another approach is to identify components of the noclcat- splicing apparatus

TEM Vol. 8, No, 10, 1997

ct al. (1997)

to t~’st the efl~, m of tissue differ-entiation o] hot-monal ;ignals on these conlpommts The isol:,( on of splicing Iactors has bee], achieved ! \ developments in ~he preparal ion of nut car extracts (Dignalm et al. 19s3) and b} ~+tab]ishing conditions of cffici(nt in vitro plicing (Kraine~ CLal. 1984). I rom this, t let-eis much evidence that the mechanis; ~s of splicing arc regw lated b} phosph(, tlation. First, the assembly of the spl .eosorne complex involves cvcles of pl ) )sphorylation and dephosph, ,rylation ( \iermoud et al. 1994). Second, several i] t Iividual proteins with regulat( lry activit} 1n splicing have been cloned. rhese incl I Je members of a f“amily of’ pi-t)teins con I,,ining multiple repeals of serimc and argii i [le residues, cal]cd SR protcim (FL~ 19$)- Manley and Tacke 1996). ‘[’he SR pt ,~eins at-e phosphorylated in vivo, as ai many other proteins invo]ve[l in splicin{. The SR proteins \vere initiall? identified Iy their common bindand

ing to a monoclinal antibody (n~ab104), the epitope of which is the phosphorylated SR domain (Roth et al. 1990). Phosphorylation is irmporlant 10SR protein function. Thus, phospholylation of the SR protein ASF/SF2 reduced its binding to nonspecific RNA and increased interactions with U 1-70K, a key protein component O( the spliceosome. Phosphotylation of SRp40 was found to be needed ft)r binding to its consensus RNA binding site, whereas dephosphorylated SRp40 bound nonspecifically (Tacke et al. 1997). Last, some protein kinases involved with the splicing apparatus have been identified (Thble 3). in addition, a pbosphatase important to splicing has recm@ been discovered by fractionation studies (Murray et al. 1997). There arc some data suggesting that SR proteins may be regulated by h(wmonal signals. The rodent version of’ I}]e SRp40 gene (cdkd HRS) was cloned by differential display experiments searching for insulin-inducible genes (Diamond et al. 1993), Insulin treatment of a ral liver cell line (H35 cells) not only induced HRS, but also incrcased phosphorylalion of SRp75, and these changes may be important to fibroncctin gene alternative splicing (Du et al. 1997). Signaling pathways may influence the Iocalimtion of’ splicing proteins. Thus, the shuttling of the abundant RNA-binding protein hnRNP A 1 between nucleus and cvtoplasrn is regulated via the MKK3/6-p38 pathway, and this is assoc iated with hyperphosphot-ylation of hnRNPAl (Lozano e( al. 1997). hnRNP A 1 has already been implicated in the selection of alternative 5‘ splice sites (Caccres et al. 1994). The regulation of pre-mRNA splicing has bum linked to the cell cycle. Progression of quiescent NIH3T3 cells in response to set-urnstimukrtion requires endogenous src kinase, and sr-c ovcrexprcssion has been shown to regulate both splicing and transport of” lyrnphokine prc-mRNAs (for example, tumor necrosis factor ~) (Ncel et al. 1995). More recent mutational analysis of SI-Chas shown that regulation of splicing requires the catalytic domain of” src, whereas a hmctional SH2 domain is needed for efi”cient exporl of partially spliced pre-nl RNAs (Pien-e and Dautry 1997). Another SR protein kinase (P1 10 PITSLRE kinase) has been identified, and this is a member of a family of kinascs related to the cell cycle regulator kinase p34cdc2 (Trembley et al. 1997). Finally, a

167-6 (!>1998,fllst~it?r Sci~n( c Inc., 1043.’ 60/98/$17.00 PlI S104.?-2760(97)00

409

Table 2. Examples of RNA sequence elements influencing alternative sdicina References Exonic enhancers Bovine GH (GGAAG, binding SR proteins) Fibronectin: ED1 exon (R-rich, binds SR) EIIIB exon (binds HRS/rat SRp40) Cardiac troponin T (GAR repeats, binds SR) Calcitonin/CGRP: Exon 4 (Dsx-type repeat and a R-rich element) R-rich element (binds 37 kD non-SR protein) Lentivirus (binds SR and lentivirus rev protein) Caldesmon (five R-rich repeats)

Exonic inhibitors a Tropomyosin

Sun et al. (1993) Lavigueur et al. (1993) Du et al. (1997) Ramchatesingh et al. (1995) Van Oers et al. (1994) Yeakley et al. (1996) Gontarek and Derse (1996) Humphrey et al. (1995)

Graham et al. (1992) and Perez et al. (1997) Del Gatto and Breathnach (1995) and Del Gatto et al. (1996)

FGF2-R

Intronic enhancers Fibronectin exon EHIB (TGCAGT repeats) Calcitonin/CGRP exon 4 (5’ ss binding U1 and SF2) FGF2-R

GABA receptor y2 c-src (binds hnRNPF) MHC-B n30 exon APP exon 8 (short enhancer and a 3 nt inhibitor)

Huh and Hynes (1994) Lou et al. (1996) Del Gatto and Breathnach (1995) and Del Gatto et al. (1997) Zhang et al. (1996) Min et al. 1995 Kawamoto (1996) Shibata et al. (1996)

Intronic inhibitors Adenovirus c-src N1 exon (CUCUCU intron repeats) (binding PTB) APP exon 8 (3 nt inhibitor) hGH hnRNP Al exon 7B (CE1 intron element binds Al)

Kanopka et al. (1996) Chan and Black (1995) Chan and Black (1997) Shibata et al. (1996) Estes et al. (1992) Chabot et al. (1997)

Dsx, Drosophila doublesex gene; 5’ SS, 5’ splice site-like sequence element; PT13, polypyrimidine tractbinding protein; R-rich, purine-rich repeats; SR, splicing proteins with serine-arginine repeat domains.

kinase of the cell cycle machinery (cyclin E or cdk2) interacts biochemically with components of the splicing machinery in eukaryotes (Seghezzi et al. 1997).

●

Problems

perhaps the greatest problem to those working on mechanisms of alternative splicing in mammals is that genetic manipulation experiments are not easy to perform. In Drosophila and in yeast, important mechanisms of however,

410

I

splicing and alternative splicing control have been elucidated and confirmed by genetic analysis. This leaves most investigators of mammalian splicing with cell culture systems and in vitro splicing experiments for testing mechanisms and with a constant question as to the relevance of any results to the situation in vivo. Additionally, hormonally responsive alternative splicing, so far, appears to be a property of differentiated cell lines, and these are liable to undergo drift and may differ in degrees of differ-

entiation. Some responses may be confined to “sublines” and may be variable even within a subline and within an experiment (Chew et al. 1995). This variability of response makes mutational analysis problematic because of the difficulty in distinguishing between an effect of a change in the sequence of a minigene from an artifact of culture conditions. A related problem is that it is often difficult to separate an effect of differentiation from a signaling effect. Some cell lines undergo differentiation and develop characteristics of a given tissue in response to manipulations of culture conditions. Thus, a change in splice choice of a gene may be attributable to the effect of differentiation (Graham et al. 1992) and only indirectly linked to a signaling system. One criterion to distinguish a direct hormonal effect from a differentiation effect is to show that a splicing switch is reversible with removal of the hormone from the culture medium (Cote and Gagel 1986, Kosaki and Webster 1993). Conversely, a splicing switch occurring in response to differentiation would be predicted to be irreversible. Also, a splicing switch occurring within a short time after stimulation is more likely to involve a direct signaling pathway (Chalfant et al. 1995). When working in vivo, it is important to be able to distinguish a change in splicing choice from a change in the rate of degradation of the mRNA isoforms (mRNA turnover). This is because there are numerous examples of hormonal stimuli changing the rate of turnover of mRNA (Ross 1995). This is particularly a problem when dealing with alternative terminal exons, because some sequences (AU-rich regions) identified as being important to the stimulated degradation of mRNAs have been localized to 3’ untranslated regions (Beelman and Parker 1995). Also, alternative terminal exons will have different polyadenylation signals, and the length of the poly(A) tail is a determinant of mRNA stability. This necessitates experiments to measure the half-lives of the alternatively spliced mRNA isoforms under the different stimulation conditions, after blocking new pre-mRNA transcription with actinomycin D or o.amantin. Where ahernative splicing of short internal exons is concerned, there is less theoretical risk of a change in mRNA turnover between two mRNA isoforms. This assumption is

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TEM Vol. 8, No. 10,1997

Table 3. Splicing and phosphorylation

Chal[an(

SR protein kinasc Clk/Sty kinase Topoisomerase I Spliceosorne assembly RNA-depencfcnt pll{.)spllol-ylation of hnRNPCl pl 10 PITLSRE phosphorylalion of RN PSI MKK3/6 p38 pathway phosphorylation of hnRNP A I Insulin induces HRS in Reubet- H3.5 CCIIS

G i et al. (1994) C will et al. (1 996) R -,si et al. (1996) hi rmoucf et al. (1994) F.,lg ct al. (1 997) T} mb]ey ct al. (1997) L( /ano et al. (1997) Di tmond et al. (1993)

CE, Mischak

H, Watson

JF., CL al.:

1995. Regu]alion o(” alternative splicing of protein kinasc Cb b> insulin. J Biol Chern 270: 13,326–1 3,332. Chan RC, Black DL: 1995. Consemecl elements

repress

splicing

intron

of a neuron-spc-

cil”ic c-src exon in vit[-o. Mol Cell Biol 15: 6377-6385. ClIan RC, Black

DL: 1997. The pol)rpyrimi

dine tract binding protein hinds upslrcam of nc([tal cell-specific c-src cxon N I to repress lhc sp] icing of the intl-on stream. Mol Cell Biol 17:4667–4676.

nol entirely secure, bowe\er, and has (c) 1X tested experimcntall~ (Lear ct al 199o) and, in the case of the insulin receptor n~RNA, still requires vcri ficat ion. Anotbcr way of assessing the cfiff’hrcnce hetwecn a change in splice choice and mRNA turnover- is to perform experiments with in titlo-tr~~nscl-i beci, racfioIabe]cd pre-nl RNA substrates in nuclear extracts. T}le lariat and lariat internlcdiates (Figure 2) ale direct pr-ociucts of’ splicing, as opposed 10 mRNA degrada1ion, and can be monitored quan titzv tivelv This necessitates the preparation of nuclear extracts capable of splicing from differentiated cell lines under- diffcrcnl simulation conditions ancl from tissues, and there are technical problems in doing tbis. The abilitv to monitor splicing in iitro is importanl ~~hen assaying the progress 01” expcr-iments attempting to isolate splicing factors. The preparation of’ ac~i\ely splicing nuclear extracts has been acbicvwf only from a small number” of cell lincx ( Eperon and Krainer 1994). These arc usually rapidlv growing rolls, in suspension cultures or loosely adherent. ProLeolysis ol’ essential splicing pr-oteins appeat-s to he one difficulty in nlaking splicing nuclear’ extrac[s from d il“l”el-entiatccl cells and tissues (La Brancl]e et al. 199 I ). %lme groLIps have atternptcd to overcome this pr-oblem by supplementing cvctracts [“mm cfif’1’erentiatedcell nuclei with actively splicing nuclear exlract (Cote et al. 1991). Transfcclion experitmenls with rnlini~enes Ienlain the most t.igorous” method of testing mechanisms in lmammalian svstems. Acfcfitionallyr, the cf’feet of ovel-cxpression 01 any splicing factor on minigene splicing ma.v be assayed in colransf’cclion experi men ts (Caccrcs et al. t 994). The genonlic stt-LlctLlreof a gene can make these experiments cfifficwlt, however, because if long in lr”onsarc

TEM Vol. 8, No. 10, 1997

clowtl-

Cbem S1., La\encfel P, Clark AJL, Ross RJM: present, Lhev can Ilake plasmid cow 1995. An alkrna(itelv spliced hulman lGF-1 stl”ucts LIllstable ar} untransfectable. transcript (lGF-IEc) lvitb hcpatic tissue exIt is n JW clear i ~,at hormone aclion pl-cssion that ciivel-ts a\v+y l“rotm tbe nlitomay inw ]Ive sever; ~ steps in RNA pro 2enic IBF.1 pcptide. Endocrinology 136: cessing, including
action.

SR splicing factors and regulates (heir iiltranuclear Clistlihution. EMBO J 15:265– ●

Ackn(~wledgmctlts

275.

The autb )t-is very : aleful to Ian Epcrx)n for rmlly disc{l >ions and much guidanc~’: to c1inic:, colleagues, Michael Bwser, Ashley Grw Inan, John Monson, and Pctcl- Trainer I ,r their support; LO Lysa Baginskv I“or ~igure 4; and to the Wellconl~’ Trust fw “L[nding.

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