Lewin Benjamin (ed.) Genes IX

Подождите немного. Документ загружается.

eral

proteins;

the U4lU6

snRNP

contains two

snRNAs and

several

proteins.

Some

proteins

are common to all

snRNP

particles.

The

snRNPs

recognize

consensus sequences.

Ul snRNA base

pairs

with the 5'

splice site,

U2 snRNA

base

pairs

with

the branch

sequence, and

U5 snRNP

acts at the 5' splice

site. When

U4 releases

U6,

the

U6 snRNAbase

pairs

with U2;

this

may

cre-

ate the catalytic center for

splicing.

An alter-

native

set of snRNPs

provides

analogous

functions for

splicing the

U12-dependent

sub-

class of introns. The

snRNA molecules

may

have

catalytic-like roles in

splicing and

other

processing

reactions.

the nucleolus,

two

groups

of snoRNAs

are responsible for

pairing

with rRNAs at

sites

that are

modified;

group

C/D snoRNAs indicate

target sites for methylation,

and

group

ACA

snoRNAs identify

sites where

uridine is con-

verted to

pseudouridine.

Splicing is usually

intramolecular,

but tr ans

splicing

(intermolecular

splicing)

occurs in try-

panosomes

and nematodes.

It involves

reaction between

a small SL RNA

and the

pre-

mRNA.

The SL RNA resembles

Ul snRNA

and

may

combine the role

providing

the exon

and the functions

of UI. In worms

there are

two types of SL RNA:

One is used for

splicing

to the 5'end

of an mRNA, and

the other is used

for

splicing to an internal

site.

Group

II introns

with nuclear introns

the use of a lariat as intermediate,

but are able

perform

the reaction

as a self-catalyzed

prop-

erty of the

RNA.

These introns

follow the GT-

AG rule, but form a

characteristic secondary

structure that

holds

the reacting

splice sites in

the appropriate apposition.

Yeast IRNA splicing involves

separate endonu-

clease and ligase reactions.

The endonuclease rec-

ognizes the secondary

(or

tertiary)

structure of

the

precursor

and cleaves both

ends of the

intron.

The two half-tRNAs releasedbyloss

the intron

can be ligated in the

presence

of ATP.

The termination

capacity

RNA

poly-

merase II has not

been characterized,

and 3'

ends of

its transcripts

are

generated

cleav-

age.

The

sequence AAUAAA, Iocated I I

to 30

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provides

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Iation. An

endonuclease and the

poly(A) poly-

merase

are associated in a complex

with other

factors that confer specificity for

the

AAUAAA

signal.

Transcription is

terminated

when

exonuclease, which binds to the 5'end

the

nascent RNA chain

created by the cleavage,

catches up

to RNA

polymerase.

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U. M., Yario, T. A.,

and Steirz,

J. A.

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processing-defective

mutations

in a

mammalian histone

pre-mRNA

are sup-

pressed

by compensatory

changes in

U7 RNA

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and in vitro.

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t709-t722.

704

Dominski,2., Erkmann,

J. A., Greenland,

J. A.,

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W. F.

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RNA binding domain

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histone

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Z. F.,

Whitfield, M. L., Ingledue,

T. C.,

Dominski, 2., and Marzluff, W.

(1996).

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the 3' end of histone

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Venema, J. and Tollervey, D.

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Balakin, A.

G., Smith,

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Caizergues-Ferrer,

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The family of box

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. Site-specific

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Review

References

705

Catalytic

RNA

CHAPTER

OUTLINE

Introduction

Group I Introns

Undertake

SeLf-SpLicing

Transesterification

The

only

factors

required

for

autospticing in vitro

group

introns

are a monovatent

cation,

a divalent cation,

and a

guanine

nucteotjde.

SpLicing

occurs by

two transesterifications,

without requir-

ing input

of energy.

The 3'-0H

end

of the

guanine

cofactor

attacks the 5'end of

the intron in

the first

transesterification-

The

3'-0H end

generated

at the end of the first

exon attacks

the

junction

between

the intron

and second exon in

the

second

transesterifi

cation.

The intron

is reteased

as a [inear motecu[e

that circutarizes

when its

3'-0H

terminus

attacks a

bond at one oftwo inter-

naI

oositions.

The

G414-A16 internal

bond of

the

intron

can

also be

attacked by

other nucteotides

a trans-splicing reaction.

Group I Introns

Form

a Characteristic

Secondary Structure

Group I introns

form

a secondary

structure with nine

duplex

reglons.

The

cores

of regions

P3, P4, P6,

and P7 have

catalytic

activity.

Regions

P4 and P7

are

both

formed

pairing

between con-

served

consensus

sequences.

sequence

adjacent

to P7

base

pairs

with

the sequence

that

contains

the reactive

Ribozymes Have

Various

Catatytic Activities

changing

the substrate

binding-site

group

I intron,

it is

possible

to introduce

alternative

sequences

that

jnter-

act with

the reactive

The reactions

ctassical enzvme

kinetics with

a low

catatytic rate.

Reactions

using 2'-0H

bonds

coutd have

been the basjs for

evolving

the

originaI

catalytic

activjties in RNA.

Some

Group I Introns

Code for Endonucleases

That

Sponsor

Mobil.ity

Mobite

introns

are abte

to insert

themselves into new

sites.

Mobi[e

group

I introns

code for

an endonuctease

that makes

a double-strand

break at

a target site.

The

intron

transposes

into

the site

of the doubte-strand

break by a DNA-mediated

repticative

mechanism.

Group Ii

Introns

May

Code for Muttifunction

Proteins

Group II

introns

can

autosplice in vitro,

but

are usualty

assisted

protein

activities

coded within

the intron.

706

A singte coding frame specifies a

protein

with reverse

tran-

scriptase activity, maturase activity, DNA-binding

motif,

and

DNA

endonuclease.

The reverse

transcriptase

generates

a DNA

copy of the RNA

sequence that transposes by a retroposon-tike

mechanism.

The

endonuclease cleaves

target

DNA

to a[[ow insertion

the transposon at a new site.

Some Autospticing Introns

Require Maturases

Autospticing

introns may require maturase

activities

encoded within the intron to assist fotding

into

the active

catatytic structure.

The Catatytic Activity

of RNAase P Is Due

to RNA

Ribonuctease P is

a ribonucteoprotein in

which the RNA has

catalytic

activity.

Viroids Have Catatytic Activity

Viroids

and virusoids form a hammerhead

structure

that has

setf-cleaving activity.

Simitar structures can be

generated

pairing

a substrate

strand

that

cteaved by an enzyme

strand.

When an

enzyme strand is introduced into

a cett, it

can

pair

with a substrate

strand target that is

then cteaved.

RNA Editing

0ccurs at

Individua[

Bases

Apo[ipoprotein-B

and

glutamate

receptors

have site-specific

deaminations catatyzed

by cytidine and adenosine

deami-

nases

that change

the coding sequence.

RNA Editing

Can Be Directed

by Guide RNAs

Extensive

RNA editing in

trypanosome mitochondria

occurs

insertions

or deletions

of uridine.

The substrate

RNA base

pairs

with

guide

RNA

on both

sides of the region

to be edited.

The

guide

RNA

provides

the

temptate for addition

(or

Less

often, deletion)

of uridines.

Editing is

catalyzed

by a complex of

endonuclease,

terminal

uridyttransferase

activity,

and RNA ligase.

Protein

Spticing Is Autocatalytic

An intein

has

the abitity to catatyze its

own removal

from

protein

in such

a way that the flanking

exteins

are

connected.

Protein

spticing is catatyzed

by the intein.

Most inteins have

two independent

activities:

protein

splic-

ing

and a homing

endonuclease.

Summary

169

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vNHr s9!

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VNUT

reopens

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primary

circle

by reacting

with the

G4r4-A16

bond.

The

UUU

(which

resembles

the

3' end of the l5-mer

released

by the

primary

ryclization)

becomes the 5'end

of the linearmol-

ecule that is formed. This

is an intefinolerularreac-

tion, and thus demonstrates

the ability

to connect

together two

different RNA molecules.

This series of reactions

demonstrates

vividly

that

the autocatalytic activity

reflects a

gener-

alized ability

of the RNA molecule

to form an

active center that can

bind

guanine

cofactors,

recognize oligonucleotides,

and

bring together

the

reacting

groups

in a

conformation that

allows bonds to

be broken and rejoined.

Other

group

I introns

have not been investigated

in as

much detail astlrre Tetrahymenaintron,

but their

properties

are

generally

similar.

The autosplicing

reaction is

intrinsic

property

of RNA invitro,

but to what

degree are

proteins

involved

invivo?

Some indications for

the involvement

proteins

are

provided

mitochondrial systems,

where splicing of

group

I introns requires

lhe trans-acting

products

other

genes.

One striking

case is

presented

t}re

qttl8

mutant of Neurospora

crassa, which is

defective in splicing

several mitochondrial

group

I introns. The

product

of this

gene

turns out to

be the mitochondrial

tyrosyl-IRNA synthetase!

This is explained

by the fact that the intron

can

take up a tRNA-like

tertiary structure

that is

stabilized by the synthetase

and which

promotes

the

catalytic

reaction.

This relationship

between the

synthetase

and splicing

consistent with

the idea that splic-

ing originated as an RNA-mediated

reaction.

subsequently assisted

RNA-binding proteins

that originally

had

other functions. The

in vitro

self

-splicing

ability may represent

the basic bio-

chemical

interaction.

The RNA

structure cre-

ates the active site,

but

able to function

efficiently in vivo

only when assisted by a

pro-

tein

complex.

Group I Introns Form

a Characteristic

Secondary

Structure

Group

I introns form

a secondary structure with

nine

duplex

regions.

The cores of

regions

P3, P4, P6,

and

P7 have

catatytic activity.

Regions

and

are both formed by

pairing

between conserved consensus seouences.

A sequence adjacent to P7 base

pairs

with the

sequence

that

contains the

reactive

3',

tla.

---oH

G-P

€"

ilili;liii.

SeLf-spticing

occurs by transesterification

reactions

in which bonds

are exchanged direct:ty.

The bonds

that

have been

generated

at each stage

are

indi-

cated by the shaded

boxes.

All

group

I introrrs can

be organized

into a char-

acteristic second,rry

structure

with

nine helices

(Pl-P9).

i i,

',i"i ',.;

shows

a model

for the sec-

ondary structure

of the

Te tr ahymen

a inlr on.

\W o

of the base-pairerl

regions

are

generated

pair-

ing between con:;erved

sequence

elements

that

are common

g|oup I introns.

P4 is constructed

from the sequences

P and

is formed from

sequences

R and

^i. The other

base-paired

regions

vary

sequenc(l

in individual

introns.

Muta-

tional analysis

ic entifies

intron

"core"

con-

taining P3, P4,

P6, and

P7, which

provides the

minimal region that

can undertake

a catalytic

reaction. The

leogths of

group I introns vary

widely, and the cr)nsensus

sequences

are

located

a considerable

distance

from

the actual

splice

junctions.

Some

of the

pairing reactions

are directly

involved in brinl;ing

the

splice

junctions

into a

conformation

that

supports

the

enzymatic

27.3 C;roup

I Introns

Form

a Characteristic

Secondary

Structure

First transfer

3'-OH

end of G attacks

5' end of

intron

s',

3',

pG-OH

pNpNpNpNpNpNpXpXFXpXpXpX

pNpNpNpNpNpN-OH

pGpXpXpXpXpXpX

Second transfer

3'-OH

of exon

attacks

end of exon

Third transfer

3'-OH end of

intron attacks

bond

bases

from 5' end

3'-OH of

attacks

pArb

5'G..UUUpA1

6CCUpU2oUG

5'G..UUUpA

at+6gg

Cyclization

Reverse cvclization

-.^e I

H,O

ZrY"-

o'\

Linearization

products

414

1.19 RNA

lrans reaction

L-15 RNA

GalapAl6CCUpUzog6

uuupAl6ccupU20uG

eron r

feoH

5, CUCUCU

First.

3'GGGAGG

lransrer

IGS

5' UAGUC 3'

AUCAG

2 bp form

at 3' end

of intron

F:{iiigI

i]"3 The

excised intron

can

form

circles by using either

two internaI

sjtes for reaction

with the

5'end, and can reopen the

circtes

by reaction with

water or otigo-nucteotides.

reaction.

Pl includes the

3'end of the left

exon.

The

sequence within

the intron

that

pairs

with

the exon

is called the IGS,

or internal

guide

sequence. (Its

name

reflects the fact

that origi-

nally

the region

immediately

3' to the IGS

sequence

shown in the figure

was thought to

pair

with

the

3'splice

junction,

thus bringing

the

two

junctions

together. This

interaction may

occur,

but does not

seem to

be essential.) A very

short sequence-sometimes

short as two

bases-between

P7 and P9

base

pairs

with the

sequence that

immediately

precedes

the

reac-

tive G

(position

4I4 in Tetrahymena)

at the 3'

end of

the intron.

The importance

of base

pairing

creating

the

necessary

core structure

in the RNA is

emphasized

by the

properties

czi-acting muta-

tions that

prevent

splicing of

group

I introns.

CHAPTER

27 Catatytic

RNA

f,tililFqil

fF,4 Group

I introns have

a common

secondary

structure that is formed

nine

base-paired regions. The

sequences of regions P4

and

are conserved,

and

iden-

tify the

jndividuat

sequence elements P,

and S.

created

pairing

between the end ofthe

left exon and

the

IGS

of the

intron;

region

between P7

and P9

pairs

with

the 3'end of the

intron.

Such mutations have been isolated for

the mito-

chondrial introns

through mutants

that cannot

remove an intron in vivo, and

they have been

isolated for the Tetrahymena intron

by transfer-

ring the splicing reaction into

a bacterial

envi-

ronment. The

construct shown in Fl{ilt*[

t3.*

allows the splicing reaction to

be followed in E.

coli. Tl;re

self

-splicing

intron

placed

at a loca-

tion that interrupts the tenth

codon of the

galactosidase

coding

sequence. The

protein

can

therefore be

successfully translated from

RNA only after the intron

has been removed.

The

synthesis of

galactosidase

in this

sys-

tem indicates

that splicing can

occur

condi-

tions

quite

distant from those

prevailing

Tetrahymena or

even

invitro.

One interpretation

of this result is that

self-splicing can

occur in

the bacterial

cell.

Another

possibility

is that

there

are bacterial

proteins

that assist

the

reaction.

Using this assay, we can introduce

muta-

tions into

the intron to see whether

they

pre-

vent

the

reaction.

Mutations

in the

group

consensus sequences that

disrupt

their base

pairing

stop splicing. The mutations

can be

reverted by making

compensating

changes

that

restore

base

pairing.

Mutations in

the corresponding

consensus

sequences in mitochondrial

group

I introns

have

770