Lewin Benjamin (ed.) Genes IX

Incubate

with

+

Gel electrophoresis

I

The

Splicing

Endonuclease

Recognizes

tRNA

o

An endonuctease

cteaves

the IRNA

precursors

at

both ends

of the

intron-

o

The

yeast

endonuclease is

a

heterotetramer

with

two

(reLated)

catatytic subunits.

r

It

uses a

measuring

mechanism

to determine

the

sites of cteavage by their

positions

retative

to a

ooint

in

the IRNA

structure.

r

The

archaeal

nuctease

has

a simpter structure

and

recognizes

a butge-hetix-butge

structuraI motif in

the substrate.

The

endonuclease is responsible

for the

speci-

ficity of intron recognition.

It

cleaves the

pre-

cursor at both ends

of the intron. The

yeast

endonuclease is

a

heterotetrameric protein.

Its

activities are illustrated

in fl{iLiH[ t*, ;|*.

The

related subunits

Sen34 and

Sen2 cleave the 3'

and 5'splice sites, respectively.

Subunit

Sen54

may determine

the sites

of cleavage by

"mea-

suring"

distance from a

point

in the IRNA

struc-

ture. This

point

is in the

elbow of the

(mature)

L-shaped

structure. The role

of subunit

Sen

I

5

is not known,

but

its

gene

is

essential in

yeast.

The

base

pair

that forms between

the first base

in

the anticodon loop

and the base

preceding

the 3' splice site is required

for

3' splice site

cleavage.

O=

Anticodon-intron

(Al)

base

pair

r*tfl*tit

llrl.iii;': The

3' and

5' cleavages

in

S. cerevisiae

pre-tRNA

are catatyzed by

different subunits ofthe endonu-

ctease. Another

subunit

may determine location

of the

cleavage

sites by

measuring distance

from the mature

structure.

The

AI base oair

is also important.

fl I

ii

iJ

F

lr

i:l

:::

.

i.'

i,i

A rc h a ea t t

R N A s

p

[i ci

n

g

e

n

do

n u clease

cleaves each strand at a bulge

in a bulge-helix-butge

motif.

An interesting insight

into the evolution

of

IRNA splicing

is

provided

by the endonucleases

of archaea. These are homodimers

or

homo-

tetramers, in which each

subunit

has an active

site

(although

only two of

the sites

function in

the tetramer) that cleaves one

of the splice sites.

The

subunit

has sequences

sequences of the active

sites

in the Sen34 and

Sen2

subunits

of the

yeast

enzyme.

The

archaeal

enzymes recognize their substrates

in

a differ-

ent

way. though.

Instead of

measuring distance

from

particular

sequences,

they recognize

a

structural feature called

the bulge-helix-bulge.

l,I:;li"illtr

:,:,.i.iii shows that

cleavage occurs

in the

two

bulges.

Thus

the origin

of splicing

of IRNA

pre-

cedes the separation

of

the archaea and

the

Fl*LJfrf

i.:{i.t.r

Spticing of

yeast

tRNA in

yifro

can be

fol

lowed by assaying

the RNA

precursor

and

products

by

gel

etectrophoresis.

26.15

The Spticing

Endonuclease

Recognizes IRNA

69t

eukaryotes. If it originated by insertion of the

intron into

tRNAs, this

must have

been

a very

ancient event.

Each 5'terminus ends

in

a

hydroxyl

group;

each

3' terminus ends

in a 2',3'-cyclic

phosphate

group.

(All

other known RNA splicing enzymes

cleave on the other side

of the

phosphate

bond.)

The two half-tRNAs base

pair

to form a

tRNA-like structure. When

AIP is

added.

the

second reaction occurs.

Both

of the unusual

ends

generated

by the endonuclease must be

altered.

The cyclic

phosphate

group

is opened to

generate

a 2'-phosphate terminus. This reac-

tion requires cyclic

phosphodiesterase

activity.

The

product

has a 2'-phosphate

group

and a

3'-OH

group.

The 5'-OH

group generated

by the

nuclease

must be

phosphorylated

to

give

a 5'-phosphate.

This

generates

a site

in

which the 3'-OH is next

to the 5'-phosphate. Covalent integrity of the

polynucleotide

chain is then restored

by

ligase

activity.

AII three activities-phosphodiesterase,

pollnucleotide

kinase, and adenylate synthetase

(which provides

the ligase function)-are

arranged in different functional domains

on

a

single

protein.

They

act sequentially to

join

the

two IRNA

halves.

The spliced molecule is now uninterrupted,

with a 5'-3'phosphate linkage at the

site of

splicing, but

it

also

has a 2'-phosphate

group

marking

the event.

The

surplus

group

must be

removed by a

phosphatase.

Generation of

a2',3'-cyclic

phosphate

also

occurs during the IRNA-splicing reaction in

plants

and mammals. The reaction in

plants

seems to be the same as in

yeast,

but the detailed

chemical

reactions are different in

mammals.

The

yeast

IRNA

precursors

also can be

spliced

in

an extract obtained from the

germi-

nal

vesicle

(nucleus)

of. Xenopus

oocytes. This

shows that the reaction is not

species-specific.

Xenopus must have enzymes able

to recognize

the introns in the

yeast

tRNAs.

The

ability to splice the

products

of IRNA

genes

is therefore

well conserved, but is likely

to

have

a different origin

from

the other

splic-

ing reactions

(such

as that of nuclear

pre-

mRNA). The

IRNA-splicing reaction

uses

cleavage and synthesis of bonds

and is deter-

mined

by sequences that are external

to the

intron.

Other splicing reactions use

transester-

ification,

in which bonds are transferred

directly,

and the sequences required for

the reaction lie

within the intron.

l@

IRNA Cleavage and

Ligation Are

Separate

Reactions

.

Release of the intron

generates

two half-tRNAs

that

pair

to form the

mature

structure.

.

The

halves have the unusual ends 5' hydroxyl and

2'-3'

cyctic

phosphate.

.

The

5'-0H end

js

phosphorytated

by a

potynucteotide

kinase, the cyclic

phosphate group

is

opened by

phosphodiesterase

to

generate

a

2'-phosphate

terminus and 3'-0H

group,

exon

ends are

joined

by an RNA ligase,

and the

2'-phosphate is removed by a

phosphatase.

The overall IRNA

splicing

reaction is

summa-

rized in

'

,r,,:ii,

.r.-:.-:ir. The

products

of cleavage

are a linear intron

and two half-tRNA

mole-

cules.

These

intermediates have unicue ends.

itl,,i::,;

Spticing of

tRNA

requires

separate

nuclease

and ligase

actjvities.

The

exon-intron boundarjes

are

cteaved by the nuclease

to

generate

2'to

3'cyctic

phosphate

and 5'0H termini. The

cyclic

phosphate

is

opened to

generate

3'-0H

and

2'phosphate

groups.

The

5'-0H

js

phosphorylated.

After

releasing the intron,

the IRNA half motecules

fotd into a

tRNA-Like

structure that now

has a 3'-0H. 5'-P

break.

This

is seated by a ligase.

Phosphodiesterase

opens

phosphate

ring

tRNA

IRNA

chain

Base

chain

Base

692 CHAPTER 26

RNA

Spticing and Processing

The

Unfolded

Protein

Response

Is

Splicing

o

Irelp

is an inner nuctear

membrane

protein

with

its N-terminaI

domain in

the ER [umen,

and

its

C-

terminal

domain in the nucteus.

.

Binding

of an unfotded

protein

to

the N-terminaI

domain

activates the

C-termjnal nuclease

by

a uto

p

hosp

horytation.

o

The

activated nuclease

cleaves

Hacl mRNA to

retease

an intron

and

generate

exons that are

tigated by a IRNA ligase.

.

The

spticed Hacl mRNA

codes for

a transcription

factor

that activates

genes

coding for

chaperones

that

hetp

to fotd unfolded

proteins.

An

unusual splicing

system that

is related to

IRNA splicing mediates

the response

to unfolded

proteins

in

yeast.

The accumulation

of unfolded

proteins

in

the lumen

of the endoplasmic

retic-

ulum

(ER)

triggers

a response

pathway

that

Ieads

to increased transcription

of

genes

coding

for

chaperones that

assist

protein

folding in the

ER. A

signal must therefore

be transmitted

from

the lumen of the ER

to the nucleus.

The sensor

that activates

the

pathway

is

the

protein

Irelp.

It is an integral

membrane

pro-

tein

(Ser/Thr)

kinase

that has

domains on

each

side of the ER membrane.

The

N-terminal

domain in the lumen

of the ER

detects the

pres-

ence

of unfolded

proteins,

presumably

by bind-

ing

to exposed motifs.

This causes

aggregation

of monomers

and activates

the C-terminal

domain on the other

side of the membrane

by

autophosphorylation.

Genes that are activated

by this

pathway

have

a common

promoter

element

called the

UPRE

(unfolded

protein

response

element). The

transcription factor Haclp

binds to

the UPRE,

and is

produced

in

response

to accumulation

ol

unfolded

proteins.

The

trigger for

production

of Haclp is the

action of Irelp

on Hacl nRNA.

The

operation of the

pathway

is summa-

rized in

; li

l;'::i

: .

.: t.

Under normal

conditions,

when the

pathway

is not

activated, Hacl mRNA

is translated into

a

protein

rhat is rapidly

degraded. The activation

of Irelp results in

the

splicing of the Hacl

mRNA to

change the

sequence

of the

protein

to a more

stable form.

This form

provides

the functional

transcription

factor that activates

genes

with the UPRE.

Unusual splicing components

are involved

in

this

reaction. IrelP has an endonuclease

activ-

ity

that acts directly on

Hacl mRNA to cleave

the two

splicing

junctions.

The

two

junctions

are ligated by the IRNA

ligase that acts

in

the

IRNA splicing

pathway.

The endonuclease

reac-

tion resembles

the

cleavage of IRNA during

splicing.

Where

does

the modification of

Hacl nRNA

occur? Irelp is

probably

located in the

inner

nuclear membrane, with the N-terminal

sensor

domain in

the

ER lumen, and the

C-terminal

kinase/nuclease domain

in the nucleus.

This

would enable

it

to

act directly on

Hacl RNA

before it is exported to

the cytoplasm.

It

also

would allow easy access

by the IRNA

ligase.

There

is no apparent

relationship between

the

Irelp nuclease activity

and the IRNA splicing

endonuclease, so

it is not obvious

how this spe-

cialized system would

have evolved.

i

ir,ritir

,,

,:,

-,'r

The unfotded

protein

response

occurs by

activating speciaI

spticing of

HACl

mRNA to

produce

a

transcription

factor that

recognizes the UPRE.

26.1.7

lhe Unfotded

Protein

Response

Is Related to IRNA

Spticing 693

i:*tJ*f

J*-.iF

When a 3' end is

generated

by termina-

tion, RNA

potymerase

and RNA are released

at a discrete

(terminator)

sequence in DNA.

F;*liftf.

iri"3t When a 3'end is

generated

by cteavage, RNA

polymerase

continues transcription while

an endonucte-

ase cteaves

at a defined sequence in

the RNA.

CHAPTER

26

RNA SpLicing

and Processing

as shown in FI*[Jftil

e*.3*.

Other RNA

poly-

merases

do

not show discrete termination,

but

continue

past

the site corresponding to the 3'

end,

which

is

generated

by cleavage of the RNA

by an

endonuclease, as shown in FfSLiRE 3S.33.

Information

about

the termination reac-

tion for eukaryotic RNA

polymerases

is less

detailed than our

knowledge

of

initiation.

RNA

polymerases

I and III have discrete termination

events

(like

bacterial

RNA

polymerase),

but it

is not clear whether RNA

polymerase

II usually

terminates in this way.

For RNA

polymerase

I, the

sole

product

of

transcription is a large

precursor

that contains

the sequences of

the major rRNA.

The

precur-

sor is subjected to extensive

processing.

Termi-

nation occurs at a discrete site

>1000

bp

downstream of the

mature f'end,

which is

gen-

erated by cleavage. Termination involves recog-

nition of an 18-base terminator

seouence bv an

ancillary factor.

With RNA

polymerase

III, transcription

in

vitro

generates

molecules with

the same 5'and

3' ends as those synthesized in vivo. The termi-

nation reaction resembles intrinsic

termination

by bacterial RNA

polymerase (see

Section I I.2I,

There Are Two

\pes

of Terminators

in E. coli).

Termination

usually occurs at the second U

within a

run

of

four

U bases, but there is het-

erogeneity, with some

molecules

ending in three

or even four U bases. The same heterogeneity

is

seen

in molecules

synthesized in vivo, so it

seems to be a bona

fide

feature

of the termina-

tion

reaction.

Just

like

the

prokaryotic

terminators,

the

U

run is

embedded

in

a G-C-rich region.

Although sequences of dyad symmetry

are

pres-

ent, they are not needed for

termination,

because mutations that abolish the

symmetry

do not

prevent

the normal completion

of RNA

synthesis. Nor are any sequences

beyond the

U run necessary because all distal

sequences can

be replaced without

any effect on termination.

The U run itself is not

sufficient for termi-

nation,

because regions of four

successive

U residues exist within transcription

units read

by RNA

polymerase

III.

(There

are no internal

U5 runs, though, which fits

with the

greater

efficiency of termination

when the terminator

is

a U5

rather

than a Ua seeuence.)

The critical

feature in termination

must therefore

be the

recognition

of a Ua sequence in a

context that

is rich in

G-C base

pairs.

How does

the termination reaction

occur?

It cannot rely

on

the

weakness of the rU-dA

RNA-DNA hybrid

region that lies

at the end of

@

The

3'

Ends

of

po[I

and

pol.III

Transcri

pts

Are

Generated by Termination

.

RNA

polymerase

I terminates transcription at an

18-base terminator sequence.

r

RNA

potymerase

III terminates transcription in

poty(U)a

sequence embedded in a G-C-rich

Seouence.

J'ends of RNAs

can be

generated

in

two ways.

Some RNA

polymerases

terminate transcrip-

tion at a defined

(terminator)

sequence

in DNA,

Promoter Terminator

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969

(the

CPE) in the

3'tail. This

is

another

AU-rich

sequence,

UUUUUAU.

In Xenopus

embryos

at least

two

type of cls-

acting

sequences

found in

the 3'tail

can trigger

deadenylation. EDEN (embryonic

deadenyla-

tion

element) is a I7-nucleotide

sequence.

ARE

elements are AU-rich

and

usually

contain

tan-

dem repeats

of AUUUA.

There

is a

poly(A)-spe-

cific RNAase

(PARN)

that

could

be involved in

the degradation.

Of

course,

deadenylation

is

not always

triggered

by specific

elements;

in

some situations

(including

the normal

degra-

dation

of

mRNA

as it

ages),

poly(A)

is degraded

unless it is

specifically

stabilized.

\cc

u

U

A

u

U

Cleavage

of the

3' End

of Histone

mRNA

May

Require

a

Sma[[ RNA

o

Histone

mRNAs

are

not

potyadenytated;

their 3'

ends are

generated

by a cleavage

reaction

that

depends on the structure

ofthe mRNA.

.

The cleavage reaction

requires

the

SLBP to bjnd to

a stem-toop

structure

and the U7

snRNA to

pair

with

an adjacent

single-stranded region.

Some mRNAs

are not

polyadenylated.

The for-

mation

of their

3' ends is

therefore

different

f rom the

coordinated

cleavage

/polyadenylation

reaction.

The

most

prominent

members

of this

mRNA

class are the mRNAs

coding

for histones

that are

synthesized

during DNA

replication.

Formation

of their

3' ends

depends upon

sec-

ondary structure.

The structure

at the

3'termi-

nus is

a highly conserved

stem-loop

structure,

with

a stem of 6

bp and

a loop of four

nucleotides.

Cleavage occurs

four

to

five

bases

downstream

of the

stem-loop. TWo

factors are

required

for the cleavage

reaction:

The stem-

loop

binding

protein

(SLBP)

recognizes

rhe

structure,

and the

U7 snRNA

pairs

with a

purine-rich

sequence

(the

histone

downstream

element,

or

HDE)

located

-10

nucleotides

down-

stream

of

the

cleavage

site.

Mutations

that

prevent

formation

of the

duplex stem of the

stem-loop

prevent

formation

of the

end of the RNA.

Secondary

mutations

that restore duplex

structure

(though

not nec-

essarily

the original sequence)

behave

as

rever-

tants. This

suggests thal

formation

of the

secondary

structure is more important

than the

exact sequence.

The

SLBP binds to

the stem-loop

and then inter-

acts

with U7 snRNP to

enhance its interaction

with the downstream

bindine

site

for

U7 snRNA.

i

ir'l':rrir

,,

;

I

":

Generation of the 3' end

of histone H3 mRNA

depends on a conserved hairpin and a sequence

that base

pairs

with U7 snRNA.

U7 snRNP is a minor snRNP consisting of

the 63

nucleotide

U7 snRNA and

a set of several

pro-

teins

(including

Sm

proteins;

see

Section 26.5,

snRNAs Are Required for Splicing).

The reaction

between

histone

H3 mRNA

and

U7 SnRNA iS drawn

in

:,,,ttiir:

,,i..

r,,,,.

The

upstream hairpin and the HDE that

pairs

with

U7 snRNA are conserved

in histone H3 mRNAs

of several

species.

The U7 snRNA

has sequences

toward its 5' end that

pair

with the

histone

mRNA consensus sequences.

3'processing

is

inhibited

by

mutations in the

HDE that reduce

ability to

pair

with U7 snRNA.

Compensatory

mutations in

U7 snRNA

that

restore comple-

mentarity

also restore

3'processing.

This

sug-

gests

that U7 snRNA

functions by base

pairing

with the histone mRNA.

The sequence of the

HDE varies among the various

histone mRNAs,

with the result that binding

of snRNA

is not

by

itself

necessarily stable,

but requires

also the

interaction with SLBP.

Cleavage to

generate

a J'terminus

occurs

at a fixed distance

from the site

recognized by

U7 snRNA, which suggests

that the snRNA

is

involved in

defining

the cleavage

site. The

fac-

tor(s) actually responsible

for cleavage,

how-

ever, have not

yet

been

identified.

Production

of

rRNA

Requires

Cleavage

Events

o

The

large and smatl

rRNAs are

reteased by cleavage

from

a common

precursor

RNA,

The major rRNAs are synthesized

as

part

of a

single

primary

transcript

that

is

processed

to

26.2I Production

of rRNA Requires

Cteavage

Events

i

:

i:

i

i.:

i: .t'

i-::.

"i

.;' Mature eu karyotic rRNAs a re

generated

by

cleavage and trimming events from

a

primary

transcript.

:

i,.i;:;ri

.::,

:::.

The

rn operons in E.

coli contain

genes

for both rRNA and

tRNA. The

exact [engths

ofthe transcripts depend on which

promoters

(P)

and

terminators

(t)

are used. Each RNA

product

must

be released from the tran-

script

by cuts

on either side.

generate

the mature

products.

The

precursor

contains the sequences

of the 185, 5.8S, and

28S

rRNAs. In higher eukaryotes,

the

precursor

is

named for its sedimentation

rate

as 45S RNA.

In lower eukaryotes

it is smaller

(35S

in

yeast).

The mature

rRNAs

are

released from the

precursor

by a combination of cleavage events

and trimming

reactions.

$ifi#ftEj ili!.:T

shows the

general

pathway

in

yeast.

There

can be varia-

tions

in

the order

of events, but basically simi-

lar reactions are involved

in

all eukaryotes.

Most

of the 5'ends are

generated

directly by a cleav-

age event. Most of the

3'ends are

generated

by

cleavage

followed by a 3'-5' trimming reaction.

Many

ribonucleases have been implicated

in

processing

rRNA, including the exosome,

which

is

an assembly

of several exonucleases

that also

participates

in nRNA degradation

(see

Section 7.13, mRNA

Degradation Involves Mul-

tiple Activities). Mutations in individual

enzymes usually do

not

prevent

processing,

which suggests

that their activities

are

redun-

dant

and that different combinations of cleav-

ages can be used

to

generate

the mature

molecules.

There are always multiple copies of the tran-

scription unit

for the rRNAs. The copies are

organized as tandem

repeats

(see

Section 6.9,

The Repeated

Genes

for rRNA Maintain

Con-

stant Sequence).

5S RNA

is

transcribed

from

separate

genes

by RNA

polymerase

III.

In

general,

the 5S

genes

are clustered, but

are separate from

the

genes

for the major rRNAs.

(In

the case of

yeast,

a 5S

gene

is associated with each major transcrip-

tion unit, but

is

transcribed

independently.)

There is a difference

in

the organization of

the

precursor

in bacteria.

The

sequence corre-

sponding to 5.8S rRNA forms the 5'end

of the

Iarge

(23S)

rRNA,

that

is,

there

is

no

process-

ing between these sequences.

Fli;ilrt*

tti-i3*

shows

that the

precursor

also contains the 5S rRNA

and one or two tRNAs. In E. coli, the seven rrn

operons

are dispersed around

the

genome;

four

rrn Loci contain one tRNA

gene

between

the

165 and 23S rRNA sequences, and the

other

rrnloci contain two IRNA

genes

in this region.

Additional IRNA

genes

may or may not

be

pres-

ent between the 55 sequence and

the 3' end.

Thus the

processing

reactions required

to release

the

products

depend on the content

of the

par-

ticular rrnlocus.

In both

prokaryotic

and eukaryotic

rRNA

processing,

ribosomal

proteins (and

possibly

other

proteins)

bind to the

precursor,

so that

698

CHAPTER 26 RNA

SpLicing and Processing

669

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