Lewin Benjamin (ed.) Genes IX

Some of the

PRP

proteins

are

components

of snRNP

particles,

but

others

function

as

inde-

pendent

factors.

One

interesting

example

is

PRPl6, a helicase that

hydrolyzes

ATP and

asso-

ciates transiently

with

the spliceosome

to

par-

ticipate

in the second

catalytic

step.

Another

example

is PRP22, another

ATP-dependent

heli-

case, which

is required

to

release

the mature

mRNA from the

spliceosome.

The conserva-

tion of bonds

during

the splicing

reaction

means

that input of

energy

is not

required

to drive

bond formation

per

se,

which implies

that the

ATP

hydrolysis

is required

for

other

purposes.

The use of

ATP by

PRPI6

and

PRP22

may be

examples

of a

more

general

phenomenon: the

use

of ATP hydrolysis

to drive

conformational

changes that

are

needed

to

proceed through

splicing.

:i,,"Lit.r-

ilii,=;

*

Spl.icing utitizes a series of base

pairing

reactions between

snRNAs and splice sites.

An extensive

mutational analysis

has

been

undertaken

in

yeast

to identify both the RNA

and

protein

components of the spliceosome.

Mutations

in

genes

needed for splicing are

iden-

tified

by the accumulation of

unspliced

precur-

sors. A series

of loci that identify

genes potentially

coding for

proteins

involved in splicing were

originally

called

RNA, but are now known

as

PRP mutants

(for pre-RNA processing).

Several

of the

products

of these

genes

have motifs that

identily them

as RNA-binding

proteins,

and

some appear

to be

family of ATP-

dependent

RNA helicases. We suppose

that, in

addition

to RNA-RNA

interactions,

protein-RNA

interactions are

important in creating or

releas-

ing structures

in the

pre-mRNA

or snRNA com-

ponents

of

the spliceosomes.

An

Alternative

SpLicing

Apparatus

Uses

Different

snRNPs

An alternative

splicing

pathway

uses

another

set

of snRNPs

that comprise

the U12

sp[iceosome'

The target

introns are

defined

by longer

consensus

sequences at

the sptice

junctions,

but usuatly

inctude the

same

GU-AG

junctions.

Some

introns have the

splice

junctions

AU-AC.

including some

that

are U1-dependent

and

some

that are U12-dependent.

GU-AG

introns

comprise

the vast

majority

(>98%

of

splicing

junctions

in the

human

genome).

Less

than

17o use

the

tions GC-AG,

and

then

there

is a

minor class

of

introns marked

by

the ends

AU-AC

(compris-

ing 0.17o of

introns).

The

first of these

introns

to be discovered

required

an

alternative

splic-

ing apparatus,

called

the

UI2

spliceosome,

which consisted

of Ul1

and

Ul2

(related

to Ul

and U2,

respectively),

a U5

variant,

and

the

U4uru.

and U6u,u.

snRNAs.

The splicing

reaction

is essentially

similar

to

that

at GU-AG

introns,

and the snRNAs

play

analogous

roles. Whether

there are

differences

in the

protein components

of

this apparatus

is not

known.

It now turns

out

that

the

dependence

on

the type of

spliceosome

is also

influenced

by

sequences

in the

intron,

so that

there

are

some

AU-AC

introns

spliced

by

U2-t1pe

spliceosomes,

and some

GU-AG

introns

spliced

by

Ul2-type

spliceosomes.

A strong

consensus

sequence

at

the left end

defines

the

Ul2-dependent

type

of

26.9

An

Alternative

Spticing

Apparatus

Uses

Different

snRNPs

681

intron:

5'GAUAUCCUUU.

.

.pyAG6

3'. In

fact,

most

Ul2-dependent

introns

have the

GU. . . .AG

termini.

In addition,

they have

a highly

conserved branch point,

UCCUUPuAPy,

which

pairs

with

U

I 2.

For

this reason,

the term

Ul2-dependent

intron is

used instead

of AU-

AC intron.

The

two types

of introns

coexist in

a vari-

ety

of

genomes,

and in some

cases

are

found

in

the

same

gene.

UI2-dependent

introns

tend

to

be flanked

by

U2-dependent

inrrons.

What is

known

about the

phylogeny

of these introns

suggests

that AU-AC

Ul2-dependent

introns

may

once have

been more

common,

but tend

ro

be converted

to GU-AG

termini,

and to

U2-

dependence,

in the

course

of evolution. The

common

evolution

of the

systems is

empha-

sized

by the fact

that

they use analogous

sets of

base

pairing

between

the snRNAs

and

with the

substrate

pre-nRNA.

The

involvement

of snRNPs in

splicing is

only

one

example

of their involvement

in

RNA

processing

reactions.

snRNPs are

required for

several

reactions

in

the

processing

of

nuclear

RNA to

mature rRNAs.

Especially

in

view of the

demonstration

that

group

I introns

are

self-

splicing,

and

that the RNA

of ribonuclease

P

has

catalytic

activity

(as

discussed

in

Chapter 27,

Catalytic

RNA),

it is

plausible

to think

that

RNA-RNA

reactions

are important

in

many

RNA

processing

events.

@

Splicing

Is

Connected

to Export

of

mRNA

r

The

REF

proteins

bind

to splicing

junctions

by

associating

with the

sp[iceosome.

.

After

spticing,

they remain

attached

to the RNA

at

the exon-exon

junction.

o

They

interact

with the

transport

protein

TAP/Mex

that

exports

the RNA through

the nuctear

pore.

After

it has

been

synthesized

and

processed,

nRNA is

exported

from

the nucleus

to the cyto-

plasm

in

the form

of a ribonucleoprotein

com-

plex.

The proteins

that are

responsible

for

transport

"shuttle"

between

the nucleus

and

cytoplasm,

remain

in

the compartment

only

briefly. TWo

important questions

are how

these

proteins

recognize

their RNA

substrates,

and

what ensures

that only

fully

processed

mRNAs

are

exported.

The

answers

in

part

may lie

in the

relative

timing

of events:

Spliceosomes

may

form

to remove

introns

before

transcription

has

CHAPTER

26

RNA

SpLicing

and Processing

been completed.

There may,

however,

also be

a direct

connection between

splicing

and export.

Introns

may

prevent

export

of mRNA

because

they are

associated

with the

splicing

apparatus. The

spliceosome

also may

provide

the initial

point

of contact

for the

export

appa-

ratus. FIGURE

t6-ie

shows a

model in

which a

protein

complex

binds to the

RNA

via the splic-

ing apparatus.

The complex

consists

of

>9 pro-

teins

and is called

the EJC

(exon

junction

complex).

The EJC is involved

in

several functions

of

splicedmRNAs.

Some

of the

proteins

of the

EJC

are

directly involved

in these

functions,

and

others recruit

additional

proteins

for

particular

functions.

The

first contact

in assembling

the

EJC is

made

with one of the

splicing

factors.

After

splicing,

the EJC remains

attached

to

the

mRNA

just

upstream

of the exon-exon

junc-

tion. The

EJC is not

associated

with RNAs

tran-

scribed from

genes

that lack

introns,

so

its

involvement

in the

process

is

unique for

spliced

products.

If introns

are

deleted from

a

gene,

its RNA

product

is exported

much

more

slowly to

the

cytoplasm. This

suggests

that the

intron may

provide

a signal for

attachment

of the

export

;

Protein

binds

I

splicing complex

I

Protein

remains

at

V

exon-exon

junction

on-exon

I

V

EJC

binds

proteins

involved

in RNA

export, localization,

decay

Fn$[.sftf

fS.ld

The EJC (exonjunction

comptex)

binds

to

RNA

by recognizing

the

spticing

comp[ex.

rl.l-;:ili;

,:i 1:

A REF

protein

binds to a splicing

factor

and

remains with

the spticed RNA

product.

REF binds to

an export

factor that

bjnds to the

nuctear

pore.

apparatus. We can now account for this

phe-

nomenon in terms of a series

of

protein

inter-

actions, as shown

in

iii;:iiii.,i1,.:,'.

The EJC

includes a

group

of

proteins

called the REF fam-

ily

(the

best characterized

member

is called

Aly).

The REF

proteins

in turn interact

with

a trans-

port protein (variously

called

TAP

and

Mex),

which

has

direct

responsibility

for interaction

with the nuclear

pore.

A similar system

may

be used to

identify

a

spliced

RNA so that nonsense mutations

prior

to the

last

exon trigger

its

degradation

in

the

cytoplasm

(see

Section

7.14,

Nonsense

Muta-

tions Trigger a Surveillance System).

Introns in

protein-coding

genes

(in

fact,

in

all

genes

except

nuclear IRNA-coding

genes)

can

be divided

into three

general classes. Nuclear

pre-nRNA

introns are

identified

only by the

possession

of the

GU .

. . AG dinucleotides

at

the 5' and 3' ends and

the branch

site/pyrimi-

dine tract

near the 3'

end.

They do not show

any common features

of secondary

structure.

Group I and

group II introns

are found

in

organelles

and in bacteria.

(Group

I introns are

found also

in the nucleus

in

lower eukaryotes.)

Group I and

group II introns

are classified

according to their

internal

organization.

Each

can be

folded into a typical

type of secondary

structure.

The

group I

and

group

II introns

have

the

remarkable

ability

to excise

themselves

from

an RNA. This

is called

autosplicing.

Group

I

introns are more common

than

group

II introns.

There is little

relationship

between

the two

classes. but

in each

case the

RNA

can

perform

the splicing

reaction

in vitro

by itself,

without

requiring enzymatic

activities

provided by

pro-

teins; however,

proteins are almost

certainly

required invivo ro assist

with

folding

(see

Chap-

ter 27, Catalytic

RNA).

r:ir,r.;

riir

'I

irr 5[6*t

that three

classes

of

introns

are

excised

by two

successive

transes-

terifications

(shown previously

for nuclear

introns in Figure

26.61.It

the

first reaction,

the

5' exon-intron

junction

is attacked

by a

free

hydroxyl

group

(provided by an

internal

2'-OH

position

in nuclear and

group

II

introns, and

by

a free

guanine

nucleotide

in

group I introns).

In

the second

reaction,

the

free

l'-OH at

the end

of the

released exon

in turn attacks

the 3'

intron-exon

junction.

There are

parallels

between

group II introns

and

pre-mRNA

splicing.

Group

II mitochon-

drial introns

are excised

by

the same

mecha-

nism as nuclear

pre-mRNAs via

a

lariat that

is

held togetherby

a

5'-2'bond.

An example

of a

lariat

produced by splicing

a

group II intron

is

shown in

i ::,r1r,,

,'ir.. i

i.

When

an

isolated

group

II RNA is incubated

in vitro

in the

absence

of

additional

components,

it is able

to

perform

the

splicing

reaction.

This means

that

the two

trans-

esterification

reactions

shown

in Figure

26.18

can

be

performed

by

the

group II intron

RNA

sequence

itself

. The

number

of

phosphodiester

bonds

is

conserved

in the

reaction,

and as

a

result an

external

supply

of

energy

is

not

required; this

could

have

been

an important

feature in the

evolution

of

splicing.

A

group

II intron

forms

into a secondary

structure

that contains

several

domains

formed

@

Group

II

Introns

Autosplice

via

Lariat

Formation

Group

II introns excjse themsetves from RNA by an

autocatatytic

splicing event.

The

splice

junctions

and mechanism

of spLicing

of

group

II introns are sjmitar to spLicing of nuctear

i ntro ns.

A

group

II intron folds into

a secondary

structure

that

generates

a catatytic site resembting the

structure of U6-U2-nuctear

intron.

26.11

Group

Ii Introns

Autosptice

via Lariat

Formation

Three

c[asses of spticing reactions

proceed

by two transesterifications.

First,

a free

0H

group

attacks

the

exon 1-intron

junction.

Second, the

0H created at the

end of

exon 1 attacks

the intron-exon

2

iunction.

by base-paired

stems

and single-stranded

loops.

Domain

5

is

separated

by twct

bases from

domain

6, which

contains

an A residue

that

donates the

2'-OEgroup

lor

the first transesterification.

This

constitutes

a

catalytic domain

in

the RNA.

compares

this secondary

structure

with the

structure

formed

by the

cclmbinarion

of U6

with U2

and of U2

with the

branch site.

The

similarity

suggests

that

U6 may have

a cat-

alytic role.

The

features

of

group

II

splicing

suggest

that

splicing

evcllved from

an autocatalytic

reacrion

undertaken

by an individual

RNA

molecule,

in

which

it accomplished

a controlled

deletion

of

an internal

sequence.

It is likely

that such a

',

Splicing reteases

a mitochondrial

group

iI intron

in the form

of a stable [ariat. Reproduced

from

Van

der Veen, R., et aL. EMB]

J. 1,987.

6:

L079-1,084.

Photo

courtesy of Leslie A.

Grivelt, European

Molecular

Biol.ogy

0rga

nisation.

reaction

requires

the

RNA

to fold into

a

specific

conlormation,

or series of conformations,

and

would

occur exclusively in

cis conformation.

The ability

of

group

II introns

to remove

themselves by an

autocatalytic

splicing

event

stands in

great

contrast to

the requirement

oi

nuclear introns

for a

complex splicing

appa-

ratus.

We may regard

the snRNAs

of

the

spliceosome as

compensating for

the lack

of

sequence information

in

the intron,

and

pro-

viding

the information required

to form

par-

ticular structures in

RNA. The functions

of the

snRNAs may have

evolved from

the

original

autocatalytic

system. These

snRNAs

actiri trans

upon

the substrate

pre-mRNA;

we

might imag-

ine

that the

ability of Ul

to

pair

with

the 5'

splice

site, or

of U2 to

pair

with

the

branch

sequence, replaced

a similar

reaction

that

required

the relevant

sequence

to

be carried

by the intron. Thus

the

snRNAs may

undergo

reactions

with the

pre-mRNA

substrate,

and

with one another,

that have

substituted

for

the series

of conformational

changes

that

occur

in RNAs

that splice

by

group

II

mechanisms.

In

effect.

these changes have

relieved

the

sub-

strate

pre-nRNA

of the obiigation

to

carry the

sequences needed

to sponsor

the reaction.

As

the splicing

apparatus

has

become

more

com-

plex (and

as

the number

of

potential

substrates

has

increased), proteins

have

played

a more

important

role.

684

CHAPTER

26 RNA

Spticing

and Processing

Nuclear

splicing constructs an

active site

from

pairing

between

U6-U2 and U2-intron

3'Exon2GA ACAAUCAU

OH

I

v

Exon 1---G

U

Group

ll splicing constructs

an active center

from the base

paired

regrons

of domains 5 and 6

Domain

5

nnnn nn

3'

Exon2 YA

YYYYAYY

OH

Domain 6

I

v

Exon 1--G

U

i:lil:..i+ir

ii:..ii:

Nuclear

spticing and

group

II spticing

invotve the

formation

of similar secondary structures. The

sequences are

more

specific in nuclear spl.icing;

group

II

spticing uses

positions

that may be occupied by either

purine

(R)

or

pyrimidine

(Y).

When an interrupted

gene

is transcribed

into

an

RNA that

gives

rise to

a single type

of spliced

mRNA, there

is no ambiguity

in assignment

of

exons

and introns.

The RNAs of

some

genes,

however,

follow

patterns

of

alternative

splic-

ing, which occurs when

a single

gene

gives

rise

to

more

than one

mRNA

sequence.

In some

cases, the ultimate

pattern

of expression

is dic-

tated by the

primary transcript,

because the

use

of different startpoints

or the

generation

of alter-

native

3'ends

alters the

pattern

of splicing.

In

other cases, a single

primary transcript

is spliced

in more than one way,

and

internal exons

are

substituted.

added, or

deleted.

In some cases,

the multiple

products

all are

made

in the same

cell, but

in others the

process

is regulated

so

that

particular

splicing

pattems

occur

only under

particular

conditions.

One of

the most

pressing

questions in splic-

ing is

to determine

what

controls

the use

of such

alternative

pathways. Proteins

that intervene

to bias

the use of

alternative

splice

sites have

been identified

in two

ways. In some

mam-

malian

systems,

it

has been

possible to charac-

terize alternative

splicing

invitro,

and to

identify

proteins

that are

required

for the

process. In D.

melanogaster, aberrations

in alternative

splicing

may be caused

either by

mutations

in the

genes

that

are alternatively

spliced

or

in the

genes

whose

products

are

necessary

for the

reaction.

irit,i,i{;;r

:ri.:.i l shows

examples

of alternative

splicing in which

one splice

site

remains

con-

stant, but

the other

varies. The

large T/small

t

antigens of SV40

and

the

products

of

the ade-

novirus EIA region

are

generated by connect-

ing a

varying 5' site to

a constant

3' site.

In the

case

of the T/t antigens,

the

5'

site

used

for T

antigen

removes a

termination

codon

that

is

present

in the

t antigen

mRNA,

so that

T anti-

gen

is larger than

t antigen.

In the

case of

the

EIA transcripts,

one of

the

5' sites connects

to

the

last exon

in

a

different

reading

frame,

again

making a significant

change

in the C-terminal

part

of

the

protein. In these

examples,

all

the rel-

evant splicing

events

take

place

in

every cell

in

which

the

gene is expressed,

so

that all

the

pro-

tein

products

are made.

There are differences

in the

ratios of

T/t

antigens in different

cell types.

A

protein

extracted from cells

that

produce

relatively

more

small t antigen

can

cause

preferential

produc-

tion of small

t RNA

in extracts

from

other

cell

types. This

protein,

which

was

called

ASF

(alter-

native splicing

factor),

turns

out

to be the

same

DifferentiaL

Use

Junctions

Alternative Spl'icing

Involves

of Sp[ice

.

Specific

exons may be excluded or inctuded in the

RNA

product

by using or faiting to use

a

pair

of

spticing

junctions.

r

Exons

may

be extended by changing one of the

splice

junctions

to use

an alternative

junction.

.

Sex determination

in Drosonhilo invotves a

series

of atternative

spLicing events in

genes

coding

for

successive

products

of a

pathway.

r

P etements of Drosophila show

germtine-specific

atternative splicing.

26.12

ALternative

Splicing

Invotves

Differential

Use of

Splice

Junctions

685

,

';-:r:!:

: i: . ; Atternative

forms

of spticing may

generate

a variety

of

protein products

from an individuat

gene.

Chang-

ing

the

sptice sites may introduce

termination

codons

(shown

by asterisks)

or change reading

frames.

as

the splicing factor

SF2,

which is required for

early steps in

spliceosome

assembly

and

for

the

first

cleavage-ligation

reaction

(see

Figure

26.13).

ASF/SF2

is an RNA-binding prorein

in

the SR family.

When

a

pre-mRNA

has more

than

one 5'splice

site

preceding

a

single 3'splice

site, increased

concentrations

of ASF/SF2

pro-

mote

use

of the 5'site nearest

to the 3'site at

the

expense

of the other

site. This

effect of

ASF/SF2

can be counteracted

by another

splic-

ing factor,

SF5.

The

exact molecular

roles

of the factors

in

controlling

splice

utilization

are not

yet

known,

but

we see in

general

terms that

alternative

splicing

involving

different

5'sites

may be influ-

enced

by

proteins

involved

in spliceosome

assembly.

In

the case

of

T/t

antigens,

the effect

probably

rests

on

increased

binding

of the SR

proteins

to the

site that is

preferentially

used.

Alternative

splicing also

may be influenced

by

repression

of one

site. Exons

2 and

3 of the

mouse

troponin

T

gene

are mutually

exclusive;

exon

2 is

used in smooth muscle,

whereas exon

3 is used in other tissues.

Smooth muscle

con-

tains

proteins

that bind to repeated

elements

Iocated on either

side of exon 3, and

which

pre-

vent

use of the 3'and 5'sites that

are needed

to include it.

The

pathway

of sex determination

in

D. melanogasler involves interactions

between a

series of

genes

in which alternative

splicing

events distinguish male and female.

The

path-

way takes the form illustrated

in F3*l"i$til

3*.If,

in which

the

ratio

of X chromosomes

to auto-

somes determines the

expression

of sxl, and

changes in

expression are

passed

sequentially

through the other

genes

to dsx, tll'e last in

the

pathway.

The

pathway

starts with sex-specific

splic-

ing

of sxl. Exon 3 of the sxl

gene

contains

a ter-

mination

codon that

prevents

synthesis

of

functional

protein.

This

exon is included

in the

mRNA

produced

in

males, but is

skipped in

females.

(Exon

skipping is illustrated

for another

example in FIfi$R{ ;S.:ri.)

As a result,

only

females

produce

Sxl

protein.

The

protein

has

a

concentration of basic amino

acids

that resem-

bles other RNA-binding

proteins.

The

presence

of Sxl

protein

changes the

splicing

of lhe transformer

(tra)

gene.

Figure 26.21

shows that this involves

splicing

a constant

5' site to

alternative J' sites.

One splicing

pat-

tern occurs in both males

and females,

and

results in an RNA

that has an

early termination

codon. The

presence

of Sxl

protein

inhibits

usage

of the normal

3' splice site

by binding

to the

polypyrimidine

tract at its

branch

site.

When

this site is skipped,

the next 3'site

is used.

This

generates

a female-specific

mRNA

that

codes

for a

protein.

Thus

/ra

produces

a

protein

only in females;

this

protein

is a

splicing regulator.

tra2 has

a

similar function in

females

(but

is

also

expressed

in

the male

germline).

The TIa

and Tra2

pro-

teins are

SR splicing factors

that

act directly

upon the target

transcripts. TIa

and TIa2

coop-

erate

(in

females)

to affect the

splicing

of dsx.

Figure

26.23

shows examples

of

cases in

which splice

sites are used

to add

or to substi-

tute

exons or introns,

again

with the

conse-

quence

that

different

protein

products

are

generated.

In the doublesex

(dsx)

gene,

females

splice the 5'

site of intron

3 to the

3'site

of that

intron;

as

a

result

translation

terminates

at the

end of

exon 4. Males

splice the

5'site

of intron

3

directly to

the 3'site

of

intron

4,

thus

omit-

ting exon

4 from the mRNA

and

allowing

trans-

686

CHAPTER

26 RNA

SpLicing

and Processing

lation

to continue through

exon 6. The result

of the alternative

splicing is

that different

pro-

teins are

produced

in

each sex: The male

prod-

uct blocks

female

sexual differentiation,

whereas

the

female

product

represses

expression of male-

specific

genes.

Alternative splicing

of dsx RNA is controlled

by competition between 3'

splice sites. /sx RNA

has an element downstream

of the leftmost

3'

splice site that

is

bound by Tra2; Tfa

and SR

pro-

teins associate with Tra2

at the site, which

becomes an enhancer

that assists binding

of

U2AF at the adjacent

pyrimidine

tract. This

com-

mits the formation of the

spliceosome to use this

3'

site

in females

rather than the

alternative 3'

site. The

proteins

recognize

the enhancer coop-

eratively,

possibly

relying on formation

of some

secondary structure as well

as sequence

per

se.

Sex determination

therefore has a

pleasing

symmetry:

The

pathway

starts

with a

female-

specific splicing event that

causes omission of an

exon that has a

termination codon, and ends

with a

female-specific

splicing event

that causes

inclusion of an exon that has

a termination

codon.

The

events have different molecurar

bases. At the first control

point,

Sxl inhibits the

default splicing

pattern.

At the last

control

point.

Tta and Tra2 cooperate to

promote

the

female-

specific splice.

The Tra and Tra2

proteins

are not needed

for normal splicing, because in

their absence

flies develop normally

(as

males). As

specific

regulators, they need not

necessarily

partici-

pate

in

the

mechanics

of the splicing reaction;

in this respect they differ from

SF2,

which is a

factor required for

general

splicing, but can also

influence

choice of alternative splice sites.

P elements of D. melanogaster

show a tissue-

specific splicing

pattern.

In somatic cells there

are two splicing events, but in

germline

an addi-

tional splicing event removes

another

intron.

A

termination codon lies in the

germline-

specific intron; as a result, a longer

protein (with

different

properties)

is

produced

in

germline.

We discuss the consequences for

control of trans-

position

in Section 2l .I5

,

P Elements Are Acti-

vated

in the Germline, and note for now that

the tissue specificity

results

from differences

in

the splicing appa ratus.

The default splicing

pathway

of the P ele-

ment

pre-mRNA

when the RNA is subjected to

a heterologous

(human)

splicing extract is the

germline pattern,

in

which

intron

3

is

excised.

Extracts of somatic cells of

D.

melanogaster,how-

ever, contain

a

protein

that inhibits

excision

of

{:lili:iiil

;i,:].::;.:'

Sex determination

in D. melonogoster

invotves

a

pathway

in

which different

spticing events

occur

in

females. Btocks

at any stage

of the

pathway

resu[t in ma[e development.

::ii.''i.iitir

":+

,i t

Alternative

splicing

events

that

involve

both

sites may cause

exons

to be

added

or substituted.

Low

\6

<-

"ehun

^

.o0/tcinn

v\

No

product

<-

Default

splicing

no

product

+

Default splicing

no

product

-

n

-

I

V

Blocks

female

differentiation

(and

promotes

male development)

I

V

Male

Tra

protein

+

I

promotes

t

-splicing

26.L2

Alternative

Spticing

Invotves

DifferentiaI

Use

of Sptice

Junctions

687

this intron.

The

protein

binds to sequences in

exon

3; if these sequences

are deleted, the intron

is

excised. The function

of the

protein

is

there-

fore probably

to repress

association of the

spliceosome

with the 5'site

of intron 3.

@

frans-Splicing

Reactions

Use

Small. RNAs

.

Spticing reactions

usuatly occur

onty in crs

between splice

junctions

on the

same motecule

of RNA.

o

frons-splicing

occurs

in

trypanosomes

and worms

where

a short

sequence

(SL

RNA) is

spticed to the

5'ends

of

many

precursor

mRNAs.

o

SL RNA has

a structure resembting

the Sm-binding

site of U snRNAs

and

may

play

an

anatogous rote

in the reaction.

In both

mechanistic

and evolutionary

terms,

splicing has

been

viewed

as an intramolecular

reaction,

essentially

amounting

to a controlled

deletion

of the intron

sequences

at the level

of

RNA.

In

genetic

terms,

splicing occurs

only

in

cis. This means

that

only sequences

on the same

molecule

of RNA

can be spliced

together. The

upper

part

of

FISU*E

3s"t4 shows the normal

situation.

The introns

can be removed from

each RNA

molecule, allowing the exons

of that RNA mol-

ecule

to be spliced together, but there isno

inter-

molecular splicing of exons

between different

RNA molecules. We

cannot say that trans

splic-

ing never

occurs between

pre-mRNA

transcripts

of the same

gene,

but we know

that it must be

exceedingly rare,

because

if

it were

prevalent

the

exons of a

gene

would

be

able to complement

one another

genetically

instead

of belonging to

a single

complementation

group.

Some manipulations can

generate

trans-

splicing. In the example illustrated

in the lower

part

of Figure 26.24,

complementary

sequences

were introduced into

the

introns

of

two RNAs.

Base

pairing

between the

complements

should

create an H-shaped molecule.

This molecule

could be

spliced

in

cli, to connect

exons that are

covalently

connected by an intron,

or it could

be

spliced

in

trans, to connect

exons of

the

juxta-

posed

RNA molecules.

Both reactions

occur ir

vitro

Another

situation

in

which trans-splicingis

possible

invitro

occurs when substrate

RNAs are

provided

in the form of

one containing a 5'splice

site and the

other containing

a 3' splice site

fllStJfts

fr*.f*i. Spticing

usuatly

occurs onty in os

between exons

carried on the

same

physical.

RNA molecute.

but

frons-spticing

can occur when

speciaI

constructs

are

made

that support

base

pairing

between introns.

CHAPTER 26 RNA

Spticing

and Processing

688

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