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releases fragments

that. may

have

different sus-

ceptibilities to exonucleases.

A region of

sec-

ondary

structure withirr

the mRNA may

provide

an obstacle

to the exonuclease,

thus

protecting

the

regions

on its 5'side.

The stability

of each

mRNA is therefore

determined

by the suscep-

tibility

its

particular

sequence

to both

endo-

and exonucleolytic

cleavages.

There

are

-12

ribonucleases

in E. coli.

Mutants

in the endoribonucleases (except

ribonuclease I,

which is without

effect) accu-

mulate

unprocessed

precursors

to rRNA

and

IRNA,

but are viable. Mutants

in the exonu-

cleases often have apparently

unaltered

phe-

notypes,

which suggests that

one enzyme can

substitute for the abse

nce of another. Mutants

lacking multiple

enzymes

sometimes are

inviable.

RNAase E is

the key enzyme in initiating

cleavage of mRNA. It nay

be the enzyme that

makes the first

cleavage for many mRNAs.

Bacterial mutants

that have

a defective

ribonuclease

E have increased

stability

(two-

to threefold) of mRNA.

However, this is not

its

only

function.

RNAase E

was originally

discovered as

the enzyme that is responsible

for

processing

5'rRNA. from

the

primary

tran-

script

specific enLdonucleolytic

process-

lng

event.

The

process

of degradation may be

cat-

alyzed by a

multienzpne

complex

(sometimes

called the degradosome)

that includes ribonu-

clease

E, PNPase,

and a helicase. RNAase

plays

dual roles. Its

N-terminal domain

pro-

vides an endonuclease

activity. The C-termi-

nal

domain

provides

a scaffold

that

holds

together the other components.

The helicase

unwinds the substrate RNA

make

it avail-

able to PNPase. According

to this model.

RNAase E makes the initial

cut and then

passes

the fragments to the

other components of the

complex

for

processin g.

Polyadenylation may

play

a role in initiat-

ing

degradation of some mRNAs in bacteria.

Poly(A)

polymerase

is associated

with ribosomes

inE.

coli, and short

(10

to 40 nucleotide)

stretches

poly(A)

are added to at least

some

mRNAs.

Triple mutations

thal

remove

poly(A) poly-

merase, ribonuclease E, and

polyrucleotide phos-

phorylase (PNPase

a 3'-5' exonuclease) have

a strong effect on stability.

(Mutations

in indi-

vidual

genes

pairs

genes

have only a weak

effect.)

Poly(A)

polymerase

may create a

poly(A)

tail that acts as a binding site for the nucleases.

The role

poly(A)

lbacteria

would therefore

be different

from

that in eukaryotic cells.

ii.,,ir'i

r,.

Degradation

of bacteriaI

mRNA

is a two-stage

process.

Endonucteolytic

cleavages

proceed

5'-3' behind

the ribosomes. The

released

fragments are

degraded by

exonucteases that

move 3'-5'.

Translation

-?-

,-\.r'

-'- . r-\'a

.-''

mRNA Stabitity

Depends

Its Structure

and Sequence

The modifications

at both ends

mRNA

protect

against degradation

by exonucteases.

Specific sequences

within

mRNA may

have

stabitizing or

destabi lizi

ng effects.

Destabitization

may be triggered

loss of

poty(A).

The major features

nRNA that

affect

its sta-

bility are summarized

i I

l'r.i

, :,r

Both struc-

ture and sequence

are important.

The 5'and

terminal structures

protect

against

degradation,

and specific sequences

within

the

mRNA may

either

serve as targets

trigger

degradation

may

protect

against

degradation:

The

modifications

the 5'and

3' ends

of mRNA

play

important

role

7.12

mRNA Stabitity

Depends on

lts Structure

and

Sequence

147

Cap

protects

Nonsense

Endonuclease

Poly(A)

protects

against

5'-3'

codons

attacks

against 3'-5'

exonuclease triggers

destabilizing

exonuclease

urveillance

sequence

5' UTR

Coding region

3'UTR

FI{UR[

?.li

The

terminaI modifications

of mRNA

protect

against degrada-

tion. InternaI

sequences

may

activate degradation systems.

Fl{,UR[

?.2?

An ARE in

a 3'nontranslated reqion

initi-

ates deqradation

of mRNA.

IRE-binding

protein

binds IRS

in absence

iroh

{4}$"

IRE-binding protein

dissociates in

presence

of iron

(A)n

f{*liRil

?,fi

IRE in a 3'nontranstated

region

controls

rRNA stabitity.

CHAPTER 7

Messenger

RNA

preventing

exonuclease attack. The

cap

prevents

5'-3' exonucleases from

attack-

ing

the

5'end,

and the

poly(A) prevents

3'-5' exonucleases from

attacking the

J'end.

Specific sequence elements within

the

mRNA may stabilize or

destabilize it.

The most common location

for destabi-

lizing

elements is within the 3'untrans-

lated region. The

presence

of such

element shortens the lifetime

of the

mRNA.

Within the coding region, mutations

that create termination

codons trigger

a surveillance system that degrades

the

mRNA

(see

Section 7.14,

Nonsense Mu-

tations

Trigger

a Surveillance

System).

Destabilizing

elements have been found

several

yeast

mRNAs, although

yet

we do

not see any

common sequences or know how

they destabilize the mRNA. They

do not neces-

sarily act

directly

(by providing

targets for

endonucleases), but may function

indirectly,

perhaps

by encouraging deadenylation. The

cri-

terion

for

defining a destabilizing

sequence ele-

ment is that its introduction into

a new mRNA

may

cause

to be degraded. The removal

of an

element from an mRNA

does not necessarily

stabilize it,

suggesting that an individual

mRNA

can have more

than one destabilizing

element.

commonfeature

some

unstable mRNAs

the

presence

of an AU-rich

sequence

-50

bases

(called

the

ARE)

that is found in

the 3'

trailer region. The consensus

sequence in

the

ARE is

the

pentanucleotide

AUUUA,

repeated

several times.

FlGUftf ?.fr?

shows that the ARE

triggers destabilization by

a two-stage

process:

first

the mRNA is deadenylated,

and

then it

decays. The

deadenylation is

probably

needed

because it causes loss

of the

poly(A)-binding

protein,

whose

presence

stabilizes

the 3'region

(see

Section 7.13, nRNA

Degradation

Involves

Multiple Activities).

some cases, an mRNA

can

be stabilized

by specifically inhibiting

the function

of a

desta-

bilizing element. Tlansferrin

mRNA

contains

sequence called

the iron-responsive

element

(IRE),

which

controls the response

of the nRNA

to changes in

iron concentration.

The IRE

located in

the 3'nontranslated

region,

and con-

tains stem-loop

structures

that bind

protein

whose affinity for

the mRNA is

controlled

iron. FIGURI

7.#3

shows that binding

of the

pro-

tein to the IRE

stabilizes the nRNA

by inhibit-

ing

the

function

(unidentified)

destabilizing

sequences

in the vicinity.

This is a

general

model

742

for the stabilization

of nRNA, that is,

stability

conferred by

inhibiting

the function

of desta-

bilizing sequences.

Decapping

and degradation

occur

in large

protein

complexes,

which may

localized in

the

form

of discrete c'ytoplasmic

units called

P-bodies, to which

other enzymes involved in

mRNA

production

or noetabolism

may be local-

ized. In fact, P-bodies

may

provide

means for

sequestering any mR|[As that

are not actively

involved in translation.

r:i;,

Deadenytation

attows

decapping to

occur,

which leads to endonucleotytic

cleavage

from the 5'end.

synthesis

(see

Section

8.9,

Eukaryotes

Use a

Complex

of Many

Initiation

Factors).

The effect

PABP

decapping

allows

the 3'end

to have

an effect

in stabilizing

the

5' end.

There is also

a connection

between

the structure

the 5'

end

and degradation

at the

3'end.

The deadeny-

lase directly binds

to the

5' cap,

and this

inter-

action

is in fact

needed

for its exonucleolytic

attack on the

poly(A).

What is the

rationale

for the

connection

between

events occurring

at both

ends of

mRNA?

Perhaps it

is necessary

to ensure

that

the

mRNA is not

left in a state

(having

the struc-

ture of one

end but

not the

other)

that might

compete with

active

nRNA

for the

proteins

that

bind to the ends.

Removal of the

cap

triggers rh'e

5'-3'degra-

dation

pathway

in which

the

mRNA

is degraded

rapidly

from the

5'end, by

the

5'-3'exonucie-

ase XRNI.

The decapping

enzyme

is concen-

trated

in discrete

cytoplasmic

foci, which

may

"processing

bodies"

where

the

mRNA

is dead-

enylated and

then

degraded

after

has been

decapped.

In the second

pathway,

deadenylated

yeast

mRNAs can

be degraded

rhe

3'-5' exonucle-

ase activity of

the exosome

a complex

exonucleases.

The exosome

is also

involved

processing

precursors for

rRNAs.

The aggrega-

tion

of the individual

exonucleases

into the exo-

some complex

may enable

3'-5'exonucleolytic

activities

to be coordinately

controlled.

The exo-

some may

also degrade

fragments

mRNA

AAAATAAAAAAA

XRNl

mRNA Deqradation

InvoLves Multipl"e

Activities

Degradation of

yeast

mRNA requires

removal of the

5'cap and the 3'poty(A).

One

yeast

pathway

involves

exonucteotytic

degradation

from

5'-+3'.

Another

yeast pathway

uses

a comptex of several

exonucteases that work in

the 3'-+5'direction.

The

deadenytase of anirnal cetls

may

bind directty

to the 5'cap.

We know the most about the

degradation of

nRNA

yeast.

There

are basically two

path-

ways. Both start with removal

of the

poly(A)

tail. This is catalyzed by a specific deadenylase,

which

probably

functions

part

of a

large

pro-

tein complex.

(The

catalytic subunit

the

exonuclease Ccr4 in

yeast;

it is also the exonu-

clease

PARN in vertebrates,

which

is related

RNAase D.)

The

enzyme action is

processive-

once it has started to degrade a

particular

mRNA

substrate,

it continue:; to whittle

away

at that

mRNA, base by base.

The major degradation

pathway

summa-

rized in

,.,,ii;:-

,,;

r,.

Deadenylation at the 3'end

triggers decapping

at the 5'end. The

basis

for

this

relationship

that th:e

presence

of the PABP

(poly(A)-binding protein)

on the

poly(A)

pre-

vents the decapping enzyme from binding to

the 5'end.

PABP is released

when the

length

poly(A)

falls below l0 to I 5 residues. Decap-

ping

occurs

by cleaving the methylated base off

the 5' end to

leave

monophosphate in

the

reaction:

mTGpppX . .

--)

mTGDP +

The enzyme requires the 7-methyl

group.

Each end of the rnRNA influences events

that occur

at the other end. This is explained

by the

fact

that the

trn,o

ends of the

mRNA are

held together by the

factors involved in

protein

7.13 mRNA

Degradation

Involves

Multiple

Activities

743

Deadenylation

3'-5' exonucleolytic

degradation

Endonucleolytic

degradation

:::i:aji

.=.r=:

Deadenylation may

lead directty

to endonu-

cteol.ytic

cleavage and

exonucleotytic

cteavage

from

the

3'end(s).

i'ii-ii$:,

1-liii:

Nonsense

mutations

mav cause mRNA

be degraded.

released

by endonucleolytic

cleavage.

ri;"il.s*iil

:.tlI

shows

that

the 3'-5'

degradation

pathway

may

actually

involve

combinations

of endonucie-

olytic

and

exonucleolytic

action.

The exosome

also found

in the

nucleus,

where it

degrades

unspliced precursors

to mRNA.

Yeast

mutants lacking

either

exonucleolytic

pathway

degrade

their mRNAs

slowly,

but the

loss

of both

pathways

is lethal.

CHAPTER

7 Messenger

RNA

Nonsense Mutations

Trigger

a SurveiL[ance

System

Nonsense mutations

cause mRNA to

be degraded.

Genes coding for the degradation

system have

been found in

yeast

and worms.

Another

pathway

for

degradation is

identi-

fied

nonsense-mediated

mRNA

decay.

FiillJR{

l.I*

shows

that the introduction

of a

nonsense

mutation often leads

to increased

degradation

of the

mRNA.

As may

be expected

from

dependence on a termination

codon,

the

degradation

occurs

the cytoplasm.

It may

represent a

quality

control or surveillance

sys-

tem for removing

nonfunctional

mRNAs.

The surveillance

system has

been studied

best in

yeast

and C. elegans,

but may

also be

important

in animal

cells. For example,

during

the formation

immunoglobulins

and T cell

receptors in

cells of the immune

system,

genes

are modified

by somatic recombination

and

mutation

(see

Chapter 23, Immune

Diversity).

This

generates

a significant number

of nonfunc-

tional

genes

whose RNA

products

are disposed

by a surveillance system.

yeast,

the degradation

requires

sequence

elements

(called

DSE)

that are

downstream

the nonsense

mutation. The

simplest

possibil-

ity

would be that these

are destabilizing

ele-

ments

and that

translation suppresses

their

use.

When translation

is blocked,

however,

tne

mRNA is

stabilized. This suggests

that the

process

of degradation

is linked

to translation

of the

mRNA

or to the termination

event in some

direct way.

Genes that

are required for

the

process

have

been identified in

S, cerevisiae

(upfIocl)

and

C. ele-

gans (smgloci)

by identifying

suppressors

of non-

sense-mediated

degradation. Mutations

these

genes

stabilize aberrant

mRNAs,

but do

not

affect

the stability

most wild-t1pe

rranscriprs.

One

of these

genes

conserved

in eukaryotes

(upfl

/smg2) It codes for

an AlP-dependent

heli-

case

(an

enzyme

that unwinds

double-stranded

nucleic

acids into single

strands).

This implies

that recognition

of the mRNA

an appropri-

ate larget

for degradation

requires

change in

its

structure.

Upf linteracts

with the release

factors

(eRFI

and

eRF3) th'al

catalyze

termination,

which is

probably

how

it recognizes

the

termination

event. It may

then

"scan"

the mRNA

mov-

Wild-type mRNA

has normal

Translation

-.>

!,]**-t"-:l

Nonsense

mutation

triggers degradation

Premature

termination

Degradation

initiates

744

ing toward

the 3' end

look

for the

down-

stream sequence

elements.

In mammalian

cells,

the surveillance

sys-

tem appears

to work only

on mutations located

prior

to the last exon-in

other

words, there

must

be an intron after

the site

mutation.

This suggests that

the system requires

some

event to occur in

the nucleus,

before the introns

are

removed

by splicirrg.

One

possibility

that

proteins

attach to the

nRNA in

the

nucleus

the

exon-exon boundary

when a splicing

event

OCCUIS.

:li:..iili: .:.";

ShTJWS a

general

mOdel fOr

the operation of such

a system. This is

similar

the way

which an nRNA

may be marked

for export from the nucleus

(see

Section

26.10,

Splicing

Connected to Export

of mRNA).

Attachment of a

protein

to the exon-exon

junc-

tion creates

mark

of the event

that

persists

into the cytoplasm.

Human homologues

of the

yeast

Upf2,3

proteins

may

involved

in such

a system. They bind

specifically to mRNA

that

has

been spliced.

Eukaryotic

RNAs

Are Transported

RNA is transported

through a membrane as a

ribonucleoprotein

particte.

A[[ eukaryotic RNAs that function in

the cytoptasm

must

be exported

from

the nucteus.

tRNAs and the RNA component

of a

ribonuctease

are imported into mitochondria.

mRNAs

can travel long distances between

plant

cet[s.

A bacterium consists

of only a single compart-

ment, so all the RNAs f

unction in the same envi-

ronment in

which they are synthesized. This is

most striking in the case

mRNA,

where trans-

lation occurs simultaneously with transcription

(see

Section 7 .7,The Life Cycle

Bacterial Mes-

senger

RNA).

RNA

transported through membranes in

the variety of instances

summarized

Ii-:.ij-:

i,.

poses ,a

significant thermody-

namic

problem

to transpoil a highly negative

RNA through a hydrophobic membrane,

and

the solution

to transport the RNA

packaged

with

proteins.

In eukaryotic cel.[s, RNAs are transcribed

in the

nucleus,

but t.ranslation occurs in the

cytoplasm.

Each

typer of RNA must be trans-

ported

into the cytoplasm to

assemble

the appa-

ratus for translation. l'he rRNA assembles with

ribosomal

proteins

into immature ribosome

J:jr'ii.

iii:

surveittance

system could

have two types

of components.

Protein(s)

must bind

in the nucteus

mark the

result

of a

spticing

event. 0ther

proteins

could

bind to the mark either

the

nucteus or cytoplasm.

They

are triggered to act

to degrade

the

mRNA when ribosomes

terminate

premature[y.

irii.iiii!

i:rr RNAsaretransportedthrough

membranes'in

avarietyof

systems'

subunits

that are

the substrates

for the

trans-

port

system.

IRNA

is transported

by a specific

protein

system.

nRNA

is transported

ribonucleoprotein,

which

forms on

the RNA

transcript

in the

nucleus

(see

Chapter

26, RNA

Splicing

and Processing).

These

processes

are

common to all

eukaryotic

cells.

Many

mRNAs

are translated

in the

cytosol,

but some

are

local-

ized within

the cell by

means

of attachment

All RNA Nucleus-+

cytoplasm

IRNA Nucleus

mitochondrion

mRNA Nurse cell-+

oocyte

mRNA Anterior+posterioroocyte

mRNA Cell-+cell

All cells

--d

Many cells

7.15

Eukarvotic RNAs

Are Transported

145

cytoskeletal element.

One situation in which

localization

occurs is

when it is important for

protein product

produced

near to the site

of its incorporation into

some macromolecular

structure.

Some RNAs are made in

the nucleus,

exported

to the cytosol. and

then

imported

into

mitochondria. The mitochondria

of some

organisms

not

code for all of

the tRNAs that

are required

for

protein

synthesis

(see

Section

4.10,

Organelle

Genomes Are Circular DNAs

That

Code for

Organelle Proteins).

In these

cases,

the additional

tRNAs must be imported

from

the cytosol. The

enzyme ribonuclease

which contains

both RNA and

protein

sub-

units, is

coded by nuclear

genes,

but is found

in mitochondria

as well

as the nucleus. This

means that

the RNA must

be imported into

the mitochondria.

We know

of some

situations in which

nRNA is

even transported

between

cells. Dur-

ing

development

of the oocyte in

Drosophila,

certain mRNAs

are transported

into the egg

from

the

nurse

cells

that surround it. The nurse

cells have specialized

junctions

with the oocyte

that allow

passage

of material

needed for

early

development.

This material

includes

certain

mRNAs.

Once in

the egg, these mRNAs

take up

specific locations.

Some

simply diffuse from

the anterior

end where

they enter,

but others

are

transported

the full length

of the egg ro

the

posterior

end by a motor

attached to micro-

tubules.

The most

striking

case of transport

of mRNA

has

been found in

plants.

Movement

of indi-

vidual

nucleic

acids over long

distances

was

first

discovered

plants,

where

viral movement

proteins

help

propagate

the

viral infection

transporting

an RNA

virus

genome

through the

plasmodesmata

(connections

between cells).

Plants

also have

a defense

system, which

causes

cells to

silence an infecting

virus. This,

too, may

involve

the

spread of components

including

RNA

over long

distance

between

cells.

Now

has turned

out that

similar systems

may trans-

pofi

mRNAs

between

plant

cells.

Although the

existence

of the

systems has

been known for

some

time, it is

only recently

that

their func-

tional importance

has

been demonstrated.

This

was shown

grafting

wild-type

tomato

plants

onto

plants

that had

the dominant

mutationMe

(which

causes

a change

in the

shape of the leaf).

mRNA from

the mutant

stock was

transported

into

the leaves

of the

wild-type

graft,

where it

changed

their shape.

mRNA

Can

Specifica[[y Locatized

Yeast Ashl mRNA forms a ribonucteoorotein

that

binds to

myosin motor.

A motor

transports it along actin filaments into

the daughter bud.

It is anchored

and translated

the bud, so that

the

protein

is found

only

the bud.

An mRNA is

synthesized in the nucleus

but

translated in the cytoplasm

of a eukaryotic cell.

passes

into

the cytoplasm in the form

of a

ribonucleoprotein

particle

that is

transported

through the nuclear

pore.

Once in the cytosol,

the mRNA may

associate with ribosomes

and be

translated. The cytosol is a

crowded

place

occu-

pied

by a high concentration

proteins.

It is

not clear how freely a

polysome

can diffuse

within

the cytosol, and most mRNAs

are

prob-

ably translated in random locations,

determined

by their

point

entry into

the cytosol,

and the

distance

that they may have moved

away from

it. However, some mRNAs

are translated

at spe-

cific

sites.

This

may be accomplished

by several

mechanisms:

An mRNA may

be specifically

trans-

ported

to a site where it is

translated.

may be universally

distributed

but

degraded

at all sites except

the site of

translation.

It may

freely

diffusible

but become

trapped

at the site of translation.

One of the best characterized

cases of local-

ization

within a

cell

that of AshI

yeast.

Ashl represses

expression

of the HO

endonu-

clease in

the budding

daughter cell, with

the

result

that HO is expressed

only in the mother

cell. The

consequence is

that mating

type is

changed only in

the mother

cell

(see

Sec-

tion 19.24,

Regulation

Expression

Con-

trols Switching). The

cause of the

restriction

the

daughter cell is that all

the Ashl mRNA

transported

from the mother

cell,

where it is

made, into

the budding

daughter

cell.

Mutations in

any one of five

genes,

called

SHEL-5,

prevent

the specific localization

and cause

AshI

mRNA to

be symmetrically

distributed

both mother

and daughter

compartments.

The

proteins

Shet,

-2,

and

-l

bind Ashl

mRNA

into

ribonucleoprotein particle

that transports

the

mRNA into

the daughter

cell. fl*tJftil ;.;S

shows

the

functions

of the

proteins.

Shelp is

a myosin

(previously

identified

as Myo4),

and

She3 and

746

CHAPTER 7 Messenger

RNA

''...'

,.'r

AshL mRNA forms

a ribo-nucteoprotein

con-

taining a

myosin motor

that moves it along

an actin

fi[ament.

She2 are

proteins

that connect the myosin

to the

nRNA.

The myosin

is a motor that moves

the

mRNA along actin filarnents.

i::,

, I

i::

. :

Summarizes the overall

process.

Ashl mRNA is exported from

the

nucleus

the

form

a ribonucleoprotein.

In the cyto-

plasm

is first

bound by She2, which recog-

nizes some stem-loop secondary

structures

within the

mRNA.

Then She3 binds to She2,

after which the myosin

Shel binds. Next, the

particle

hooks on to an actin filament and moves

to the bud.

When Ashl nRNA

reaches the bud,

it is anchored there,

probably

proteins

that

bind specifically to themRNA.

Similar

principles govern

other cases where

mRNAs are transported to specific sites. The

nRNA

is recognized

by means of czs-acting

sequences, which usually are regions of sec-

ondary structure in the 3'untranslated region.

(AshI

mRNA is unusual in that

the crs-acting

regions are

the coding frame.) The mRNA is

packaged

into a ribonucleoprotein

particle.

some

cases, the transpOrted nRNA

can

be visu-

alized

in very large

particles

called

nRNA

gran-

ules. The

particles

are

large

enough

(several

times the size of a ribosome) to contain many

protein

and RNA components.

A transported

mRNP must

be connected to

motor that moves it along a system of tracks.

The tracks can be either actin filaments or micrt-r-

tubules.

Whereas Ash.t uses a myosin motor on

actin tracks. oscar

mRNA in

the Drosophila egg

uses a kinesin motor to move along micro-

tubules.

Once the mRNA reaches its destina-

tion,

it needs to be anc.hored in

order

prevent

it from diffusing

away. Less is known

about this,

r.r:.:r'

-j.

1:']

Ashl mRNA

exported

from the nucleus

into

the cytoplasm, where

assembled

into a

complex with

the She

proteins.

The complex transports

it along actin

fit-

aments to the bud.

but the

process

appears

be independent

transport.

An nRNA that

is transported

along

microtubules may anchor

actin

filaments at

its destination.

Sum

mary

Genetic

information carried

DNA

expressed

in two stages: transcription

DNA into

mRNA

and translation of

the mRNA

into

protein. Mes-

senger

RNA is transcribed

from one

strand of

DNA. It is complementary

to this

(noncoding)

strand and

identical

with

the other

(coding)

strand.

The sequence

nRNA,

in triplet codons

5'-3',

is related to the

amino

acid sequence

protein,

N- to C-terminal.

The adaptor

that

interprets

the meaning

a codon

is transfer

RNA,

which

has a compact

L-shaped tertiary

structure;

one

end of

the IRNA

has an anticodon

that

is complementary

the

codon, and

the other

end

can be

covalently

linked to the specific

amino

acid that

corre-

sponds

to the target

codon.

A IRNA

carrying

amino acid

is called

an aminoacyl-tRNA.

The ribosome

provides

the

apparatus

that

allows aminoacyl-tRNAs

to bind

to their

codons

nRNA. The small

subunit

of the

ribosome

bound

to mRNA;

the

large subunit

carries

the

nascent

polypeptide. A

ribosome

moves along

mRNA from

an initiation

site

in the

5'region

a termination

site

in the

3'region,

and

the appro-

priate

aminoaryl-tRNAs

respond to

their codons,

unloading their

amino

acids,

so that

the

grow-

ing

pollpeptide

chain

extends

by one

residue

for

each codon traversed.

The translational

apparatus

is not specific

for

tissue or organism;

nRNA

from one

source

can be translated

the

ribosomes

and

tRNAs

from another

source.

The

number

of times

any

7.17 Summary

747

mRNA is

translated is a function

of the affinity

of its initiation

site(s) for ribosomes

and its sta-

bility. There

are

some cases in which

transla-

tion of

groups

nRNA

or individual

mRNAs

specifically

prevented:

this is

called transla-

tional control.

typical mRNA

contains both a nontrans-

lated

5'leader

and a 3'trailer

as well as coding

region(s).

Bacterial mRNA

is usually

poly-

cistronic, with

nontranslated

regions

between

the

cistrons. Each

cistron is represented

by a

coding region

that starts with

a specific initia-

tion

site and

ends

with

a termination

site. Ribo-

some

subunits associate

at the initiation

site and

dissociate

at the

termination

site of each codine

reglon.

growing

E. coli

bacterium has

-20,000

ribosomes

and

-200,000

tRNAs, mostly

in the

form

of aminoacyl-IRNA.

There

are

-1500

nRNA molecules,

representing

two to three

copies

of each

of 600 different messengers.

single mRNA

can be

translated by many

ribosomes

simultaneously,

generating

polyri-

bosome

(or polysome).

Bacterial

polysomes

are

large,

typically

with tens

of ribosomes

bound

to a

single mRNA.

Eukaryotic

polysomes

are

smaller, typically

with fewer

than ten ribosomes;

each mRNA

carries

only a

single coding

sequence.

Bacterial

mRNA

has an

extremely

short

half-life

only a few minutes.

The 5'end

starts

translation

even

while the

downstream

se-

quences

are being

transcribed. Degradation

is ini-

tiated

by endonucleases

that

cut at discrete

sites,

following

the ribosomes

the 5'-l'direction,

after

which

exonucleases reduce

the

fragments

to nucleotides

by degrading

them from

the

released

end toward

the 5'

end. Individual

sequences

may

promote

or retard

degradation

in bacterial

mRNAs.

Eukaryotic

nRNA must

processed

the nucleus

before it is transported

to the cyto-

plasm

for

translation.

A methylated

cap is

added

to the 5'end.

It consists

of a nucleotide

added

to the

original end

by a 5'-5'bond,

after

which methyl groups

are added.

Most

eukary-

otic nRNA

has

-200

base

sequence of

poly(A)

added

to its 3'terminus

in the nucleus

after

transcription,

but

poly(A)-

mRNAs

appear

be translated

and

degraded

with

the same

kinetics

poly(A)+

mRNAs. Eukaryotic

mRNA

exists

ribonucleoprotein

particle;

some

cases

mRNPs

are

stored that

fail to

be trans-

lated.

Eukaryotic

mRNAs

are

usually

stable

for

several

hours.

They may

have multiple

sequences

that

initiate

degradation;

examples

are known in

which the

process

is regulated.

Yeast mRNA is degraded

(at

least) two

pathways.

Both

start

with

the removal

poly(A)

from

the 3' end, causing loss

poly(A)-

binding

protein,

which in turn leads

to removal

of the methylated cap from

the 5' end.

One

pathway

degrades the mRNA from

the 5'end

by an exonuclease. Another

pathway

degrades

from the 3' end

by the exosome, a

complex con-

taining

several exonucleases.

Nonsense-mediated

degradation leads

the

destruction of mRNAs that have

a termina-

tion

(nonsense)

codon

prior

the last exon.

T}re

upflociin

yeast

and the

smgloci in

worms

are required for the

process.

They include

helicase activity

to unwind mRNA

and a

pro-

tein that interacts

with the factors

that termi-

nate

protein

synthesis.

The features

of the

process

mammalian cells

suggest that

some

of the

proteins

attach

to the mRNA

in the

nucleus

when RNA

splicing occurs

to remove

introns.

mRNAs

can be transported

to specific

loca-

tions

within a cell

(especially

in embryonic

development). In

the AshI system

yeast,

mRNA is

transported from

the mother

cell into

the daughter cell by

myosin

motor

that moves

on actin filaments. In

plants,

mRNAs

can

transported long

distances between

cells.

References

Transfer

RNA

Forms

a Clover[eaf

Review

Soll, D. and RajBhandary,

(1995).

IRNA

Struc-

ture, Biosynthesis,

and Function.

Washington,

DC: American

Society for Microbiology.

Resea rch

Chapeville, F. et al.

(1962).

On the role

soluble

RNA in

coding

for

amino acids.

Proc

Natl.

Acad. Sci.

USA

48, 1086-1092.

Hoagland,

M. B.

et al.

(1958).

soluble RNA

intermediate

protein

synthesis.

J. Blol.

Chem.2)I, 241-257.

Holley, R.

W. et al.

(I965).

Structure

of an RNA.

Science I47, 1462-1465

Messenger

RNA Is Translated

by Ribosomes

Resea rc h

Dintzis,

H. M.

(1961).

Assembly

of the

peptide

chain of hemoglobin.

Proc

Natl. Acad.

Sci.

usA 47

247-26r.

748

CHAPTER

Messenger

RNA

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pulg

sauosoqlu

^uPW

Resea rch

Cui, Y., Hagan,

K. W., Zhang,

S., and Peltz,

S. W.

(

1995).

Identification

and characterization

genes

that are required

for the

accelerated

degradation

of mRNAs containing

prema-

ture

translational termination

codon. Genes

Dev.9, 42)-4)6.

Czaplinski, I(., Ruiz-Echevarria,

J., Paushkin,

S. V., Han,

X., Weng, Y., Perlick,

H. A., Dietz,

C.,

Ter-Avanesyan,

D., and Peltz,

S. W.

( I 998

)

. The

surveillance complex interacts

with the

translation release factors

to enhance

termination

and degrade aberrant

mRNAs.

Genes Dev. 12, 1665-1677.

Le Hir,

H., Moore,

M. J., and Maquat,

L. E.

(2000).

Pre-nRNA

splicing alters mRNP

composition:

evidence

for stable

association of

proteins

at exon-exon

junctions.

Genes

Dey.

).4,

r098-1

108.

Lykke-Andersen,

J., Shu, M. D.,

and Steirz, J. A.

(2000).

Human

Upf

proteins

target an nRNA

for

nonsense-mediated

decay when bound

downstream

of a termination

codon.

Cell lO).

I l2l-1

t 3l.

Peltz,

S. W., Brown,

A. H., and

Jacobson, A.

(1993).

mRNA

destabilization

triggered

premature

translational

termination

depends

on at least

three cls-acting

sequence

elements

and

one trans-acting f.acI-or.

Genes Dev. 7

t7)7-t7

54.

Pulak,

R. and Anderson

(19%1.

mRNA

surveil-

lance

the C. elegans

smg

genes.

Genes

Dev.7,

885-l 897

Ruiz-Echevarria,

J. et al.

(1998).Identifying

the right stop:

determining how

the surveil-

lance

complex

recognizes

and degrades

aberrant

mRNA. EMBO

J.15, 2810-28I9.

Weng, Y.,

Czaplinski, K.,

and Peltz,

(1996).

Genetic and

biochemical

characterization

mutants

the

ATPase

and helicase

regions

the Upf I

protein.

Mol Cell Biol.

16, 5477-5490.

Weng, Y.,

Czaplinski, K.,

and Pelrz,

(1996).

Identification

and characterization

muta-

tions in

the upfl

gene

that affect

the Upf

pro-

tein complex, nonsense

suppression,

but not

mRNA

turnover. Mol.

Cell Biol.

16. 549t-5506.

Eukaryotic

RNAs Are Transported

KCVICWS

Ghoshroy,

S., Lartey,

R.,

Sheng, J., and

Citovsky, V.

(1997).

Transport

proteins

and nucleic

acids

through

plasmodesma:a.

Ann. Rev. Plant. Phys-

iol.Plant Mol Biol 48,27-50.

Jansen, R. P.

(2001).

mRNA localization:

message

on the

move.

Nat. Rev. Mol.

Cell

Biol.2.

247-256.

Lucas, W. J. and Gilbertson, R. L.

(1994).

Plasmod-

esmata in relation to

viral

movement

within

leaf tissues. Ann. Rev. Phytopathol.

32,

387-4ll.

Vance,

and Vaucheret, H.

(2001).

RNA

silencing

plants-defense

and counterdefense.

Science

292,2277-2280.

Resea rch

ICm, M.,

Canio, W., I(essler, S., and

Sinha,

(2001).

Developmental changes

due to long-

distance movement of a homeobox

fusion

transcript in tomato.

Science 29), 287-289.

Puranam, R.

S. and

Attardi,

(2001).

The

RNase

associated with HeLa cell mitochondria

con-

tains an essential RNA component

identical in

sequence to that of

the

nuclear

RNase P. Mol.

Cell Biol. 2l

548-561.

mRNA

Can Be Specificatty

Localized

Reviews

Chartrand, P.,

Singer, R. H., and Long,

R. M.

(2001).

RNP localization

and transport in

yeasL.

Annu Rev.

Cell

Dev.

Biol. 17

297-310.

Jansen, R. P.

(2001

)

. mRNA

localization:

message

the

move.

Nat. Rev. Mol.

Cell Biol 2.

247-256.

ICoc, M.,

Zearfoss,

R.,

and

Etkin,

L. D.

(2002).

Mechanisms

of subcellular

mRNA localiza-

tion. Cell 108, 533-544.

Palacios, I. M.

and Johnston, D.

(2001).

Getting

the message across:

the

intracellular

localiza-

tion of mRNAs

in higher eukaryoles.

Annu.

Rev.

Cell

Dev.

Biol. 17, 569-614.

Resea rch

Bertrand,

E.,

Chartrand, P., Schaefer,

M., Shenoy,

S. M., Singer, R. H.,

and Long, R.

(1998).

Localization

of ASHI mRNA

particles

living

yeast.

Mol.

Cell 2, 4)7445.

Long, R. M.,

Singer, R. H., Meng,

X.,

Gonzalez,L,

Nasmyth, I(., and Jansen, R.

(1997).

Mating

type switching

yeast

controlled

by asym-

metric localization

of ASHI

mRNA.

Science

277,

j83-387

150

CHAPTER

7 Messenger

RNA