Retroviral RNA Processing and stability - Hellas · Retroviral RNA Processing and stability Karen Beemon Johns Hopkins University m7G gag pol env src AAA m7G env src AAA m7G src AAA

Retroviral RNA Processing and stability

Karen Beemon

Johns Hopkins University

gag pol env src AAAm7G

env src AAAm7G

src AAAm7G

Retroviruses hijack host cell gene expression machinery to generate progeny

virions

AAAAA

gag pol env src

Simple retroviruses use cis-acting RNA sequences to express unspliced RNA

NRS RSE DR2DR1

1) Stability Element (RSE) (pol): Stabilize RNA with long 3’UTR

2) Negative Regulator of Splicing (NRS) (gag): suppresses splicing, promotes pA

(pseudo-5’ SS:binds U1/U11 & SRs), promotes rapid lymphomas

3) Direct Repeat (DR) (3’UTR): Exports unspliced RNA (virion assembly) Needs

Tap and Dbp5.

.

Rous sarcoma virus RNA

Arrigo and Beemon

MCB 1988

Deletions in gag affect RSV RNA splicingand stability

SPLICING: How is the correct ratio of spliced/unspliced viral RNAs maintained?

�5’ splice site efficient, 3’ splice sites inefficient (RSV & HIV)

�Exonic splicing enhancers and silencers

�Negative regulator of splicing (NRS) sequence in gag

5’ splice site decoy, 500 nts downstream of 5’ splice site

Also promotes pA at 3’ LTR

SD SA ESE SA

AAAA

SR proteins snRNPs

hnRNP H uG/GUuuGUAUCCUUC

U11 snRNP

__________U1 snRNP

gag

NRS

AA: spl.

CC: 0uG/GUAAGUAUCCUUC

uG/GUccGUAUCCUUC

NRS

Bulged U in NRS 5’ helix promotes U1 snRNP binding

Cabello-Villegas et al. RNA 2004

(Yun-Xing Wang, NCI)

uG/GUuuGU

50S- heparinA

B18S

28S

+ heparin

NRS-Ad3’ RNA makes aberrant 50S spliceosomal complex

Giles & Beemon, MCB 2005

NRS Ad 3’

NRS sequesters 3’SS in aberrant complex

U11

GRAINGER et al. RNA 2005; 11: 533-557

U5 Prp8 is critical to spliceosome function, cross-links to 5’SS

U5 Prp8 does not X-link to the NRS 5’ splice site

Site-specific label at +1/+2 of NRS decoy 5’ splice site

Giles & Beemon, MCB 2005

hPrp8

GpU

NRS Ad

1. The NRS is a 5’ splice site decoy that inhibits

splicing and promotes polyadenylation.

2. U5 Prp8 is misplaced.

How do NRS mutant ALVs act in vivo?

Mutation at G919A (+5) converts splicing suppressor to 5’ SS

0 1 2 3 4 5 6 7 8 9 10

0

25

50

75

100

WT

G919A

Weeks

Percent Survival

ALV with NRS point mutation induces rapid-onset lymphomas

Polony et al. 2003 JVI

∆ALV c-myb

SD SA SA

NRS

Retroviral Stability Element (RSE)

Prevents Nonsense-Mediated mRNA Decay (NMD) of viral RNA with long

3’ UTR

The Rous sarcoma virus unspliced RNA must evade host translation quality

control

Long mRNA (>9 kb)

Unspliced mRNA

Polycistronic

Long 3’ UTR after the first open reading frame

Long half-life (9-20 hrs)

gag pol env src AAAm7G

RSE

Arrigo and Beemon

MCB 1988

Deletions in gag affect RSV RNA splicing and stability

PTC

U

S

RNA termination codon in gag leads to instability of unspliced RNA

Barker and Beemon, MCB 1994

Nonsense codon at nt

15

47

18

06

19

24

21

00

23

19

24

55

WT

7kb 3’ UTR

AAAA

NTC

gag

A deletion downstream of gag NTC also destabilizes RNA

WT AAAA

AAAA∆RSE

Nts 2486-2886 deleted

mock WT ∆RSE

control

experimental

NTC

NTC

100% 26%

Weil and Beemon, RNA 2006

gag

Rous Stability Element (RSE)

NTC

PTC+ + +- --

Control

Experimental

wt Mini

Mini

+RSE

% of wt100 31 80 38 73 24

Stability element is dispensable when termination occurs near the poly(A) tail

AAAAA

AAAAA

AAAAA

wt

Mini

Mini +

RSE

~650 nts400 nts

Dominant negative Upf1 stabilizes RNA lacking the RSE

Implicates nonsense-mediated mRNA decay

0

0.2

0.4

0.6

0.8

1

1.2

Dominant Negative

Upf1

PTC

RSE∆

WT

WT

Upf1

Fraction W

T

Weil and Beemon, RNA 2006

Nonsense codons are usually recognized by downstream exon-junction complexes (EJC) in mammals

Exon-junction complex (EJC) is a heterogeneous collection of proteins

eIF4AIIIeIF4AIII

Upf3

Upf2

Upf1

p15

TAP

Magoh

REF/ALYUAP56

RNPS1

SRm160

BTZ

Y14

Nonsense-mediated mRNA decay

Splicin

g

Ex

po

rtLo

caliz

atio

n

Un

cha

racte

rized

Exon1 Exon2

mRNP

-20-24 nts

Ter

Ter

Ter∆RSE

reverse

Ter

forward

Can the RSE stabilize premature termination within the gag gene?

Ter

reverse

Ter

Ter

Ter∆RSE

forward

0 1 2 4 0 1 2 4 0 1 2 4 0 1 2 4

WT Ter (1924) Ter RSE for Ter RSE rev

exp

control

Re

lati

ve

RN

A le

ve

l

actinomycin D (hrs)

0

0.5

1

1.5

0 1 2 3 4

Ter

Ter RSE For

Ter RSE Rev

WT

The Stability Elementstabilizes PTC-bearing viral RNA

Is there a stability element downstream of other viral genes?

AAA…gag env srcpol

pol

UTR

env

UTR

src

UTRRSE

C

UTR insertion into

gag

Ter

…C

WT ∆RSE

Sequence found after HIV-1 gag termination codon stabilizes termination in RSV

gag pol vif

vpr

rev

tat vpu

env tat

rev nef

??

HIV

genome

eRF 1 and 3 stabilize ∆RSE RNA, so perhapsRSE promotes termination

Fraction W

T

Control

Experimental

eRF1 + eRF3 - + - + - +

WT PTC RSE∆

∆∆

Johanna Withers

5% acrylamide 10% acrylamide

RSE migrates anomalously in denaturing gels

Jason Weil

RSE sediments as monomerin analytical ultracentrifuge

Van Moudrianakis

0 5 10 15 20 25 30 35-1.0 x 10-5

1.9 x 10-4

3.9 x 10-4

Equilibrium Velocity

Radius

Abs 260 nm

1.0

0.8

0.6

0.4

0.2

0.06.0 6.1 6.2 6.3 6.4 6.5 6.76.6

Frictional Ratio = 1.7

Sedimentation Coefficient

g(s*)

Michael Hadjithomas

SHAPE chemistry(Selective 2’-hydroxyl acylation analyzed by primer extension)

A

U

C

G

G

G

G

G

G

G G

C

C

C

C

U

U

U

U

A

A

A

A

C

+

*

*

*

*

*

*

*

*

M - +

8

17

1

10

20

A

U

C

G

G

G

G

G

G

G G

C

C

C

C

U

U

U

U

A

A

A

A

C

5’ 3’

A UCG G G GGGG G C CCC UUU UA AAAC

A UCG G G GGGG G C CCC UUU UA AAACA UCG G G GGGG G C CCC UUU UA AAAC

A UCG G G GGGG G C CCC UUU UA AAAC

A UCG G G GGGG G C CCC UUU UA AAACA UCG G G GGGG G C CCC UUU UA AAAC

5’ 3’

1M7

1M7

Kevin Weeks, UNC

Structure of the RSE

Model: UTR sequences downstream of gag NTC promote proper termination and RNA stability

AUG

UGA

UGA

NMDAUG

UGA

No

NMD

PTC NTC

NTC

Faux UTR: NMD

Proper Termination: No NMD

RSE inhibits NMD at gag TER

RSE inhibits NMD at gag TER

Does it promote translation termination?

Nuclease footprinting of ribosomes on mRNAs indicate in vivo ribosome positions

Nicholas Ingolia

Ribosome footprints reveal in vivotranslation

Ribosome Profile of RSV

CHX treated 1 hr

Summary• When RSE is deleted, RNA undergoes NMD

• RSE can convert PTC to normal stop codon

• RSE is a highly structured element

• HIV 3’UTR can stabilize an RSV PTC-may be common element in retroviruses or all mRNAs with long 3’UTRs

• Mechanism?Jason Weil

Johanna Withers

– Promote termination-interact with ribosome?

– Interact with Poly (A)/PABP?