mRNA stability by kk sahu

Introduction of mRNA & stability
Stabilization of mRNA
5’ capping
3’ polyadenylation
Destabilization of mRNA
Conclusion
References

1. mRNA STABILITY
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. Introduction of mRNA & stability
Stabilization of mRNA
5’ capping
3’ polyadenylation
Destabilization of mRNA
Conclusion
References

3. mRNA and Stability
TheextenttowhichanRNAmoleculeretainsits
structuralintegrityandresistsdegradationby
RNase,andbase-catalyzedhydrolysisunder
changinginvivoorinvitroconditions.
mRNA stabilityanddegradationareplay
importantroleinregulationoflevelofaparticular
protein.

4. Transcription & Product
(i)Polycistronic mRNA
(ii)Monocistronic mRNA

5. Stabilization of mRNA-
RNAs are made in the nucleus of a eukaryotic cell, but
function in protein synthesis in the cytoplasm. As cytoplasm
contains many nuclease proteins and Rnase that can
degrade the pre-mRNA therefore processing of mRNA is
required.
Processing involves-1. 5’ capping
2. 3’ polyadenylation

6. 1. 5’ capping

7. Caps provide at least four functions:
Protection of mRNA from degradation.
Enhancement of mRNA’s translatability
Transport of mRNA out of nucleus
Proper splicing of pre-mRNA

8. 2.3’cleavage&polyadenylation
EarlysequencingofcDNAclonesfromanimalcells
showedthatnearlyallmRNAscontainthesequence
AAUAAA10–35nucleotidesupstreamfromthepoly(A)
tail.Furthermutagenesisstudiesrevealedthata
secondsignaldownstreamfromthecleavagesiteis
requiredforefficientcleavageandpolyadenylationof
mostpre-mRNAsinanimalcells.Thisdownstream
signalisnotaspecificsequencebutratheraGU-richor
simplyaU-richregionwithin≈50nucleotidesofthe
cleavagesite.

9. A 360-kDa cleavage and polyadenylation
specificity factor(CPSF), composed of
four different polypeptides, first forms
an unstable
complex with the upstream AAUAAA
poly(A) signal.
A 200-kDa heterotrimercalled cleavage
stimulatory factor (CStF), which
interacts with the G/Urichsequence.
A 150-kDa heterotrimercalled cleavage
factor (CFI); and a second, poorly
characterized cleavage factor (CFII).
Finally, a poly(A) polymerase (PAP)
binds to the complex before
cleavage can occur. This
requirement for PAP binding links
cleavage and polyadenylation, so
that the free 3’ end generated is
rapidly polyadenylated.

10. wecancollectandgraph
dataontheaccumulationof
mRNAwithapolyAtailand
HistonemRNA,whichlacks
apolyAtail,inorderto
compare thetwo.The
turnoverofmRNAlackinga
polyAtailismuchhigher
thanmRNAcontainingapoly
Atail.PolyAmayplayarole
intranslationofmRNAby
increasingthestabilityof
mRNAandallowingmRNAto
function normally.
Exponentialequationsand
graphsmakeiteasierforthe
half-livesofmRNAtobe
calculatedandcompared.

11. mRNA degradation
TheconcentrationofanmRNAisafunctionofbothitsrateof
synthesisanditsrateofdegradation.Forthisreason,iftwo
genesaretranscribedatthesamerate,thesteady-state
concentrationofthecorrespondingmRNAthatismorestable
willbehigherthantheconcentrationoftheother.
Someproteinsineukaryoticcellsarerequiredonlyforshort
periodsoftimeandmustbeexpressedinbursts.Forexample,
certainsignalingmoleculescalledcytokineswhichare
involvedintheimmuneresponseofmammals,are
synthesizedandsecretedinshortbursts.Similarly,manyof
thetranscriptionfactorsthatregulatetheonsetoftheSphase
ofthecellcycle,suchasc-FosandcJun,aresynthesizedfor
briefperiodsonly.Expressionofsuchproteinsoccursin
shortburstsbecausetranscriptionoftheirgenescanberapidly
turnedonandoffandtheirmRNAshaveunusuallyshorthalf-
lives,ontheorderof30minutesorless.

12. mRNA Decay Pathways
Deadenylation-dependent pathways
Deadenylation-independent pathways:
1. Endoribonucleolytic decay
2. Nonsense-mediated decay (NMD)

13. Deadenylation-dependent mRNA Decay
WhenmRNAprocessingiscomplete,the
mRNAbearsa5'capstructureand3'poly(A)
tailthatprotectthemessage from
exonucleolyticdecay.Thefirststepinthe
decayofmostwild-typemRNAsisshortening
ofthepoly(A)tailbyadeadenylase.Once
poly(A)shorteningiscomplete,the5'7-
methylguanosinecapisrapidlyremovedand
therestofthemRNAisattackedby5'and3'
exonucleases.

15. Endoribonucleolytic Decay
There are a few messenger RNAs that
degrade by a minor pathway known as
endoribonucleolytic decay.
Endoribonucleases recognize specific
sequence elements within the transcript and
cleave the mRNA internally. The cleavage
event generates free 3' and 5' ends that are
easily accessible to exonucleases and the
products of the cleavage reaction are
therefore rapidly degraded.
In contrast, stabilizer protein may block the
binding of endoribonuclease.

17. Interestingly, several mRNAs that are
degraded by endoribonucleolytic decayalso
interact with stabilizer proteins that block
access of the endoribonuclease to its
cleavage site.
e.g., an endonuclease from Xenopus laevis
hepatocytes, PMR1, can cleave the
vitellogenin mRNA but its action is prevented
by binding of the vigilin protein to a site that
overlaps the PMR1cleavage site.
Similarly, the a-globin mRNA is cleaved at a
site in its 3' UTR by an erythroid-enriched
endonuclease. In this case, cleavage is
inhibited by binding of the a-CP complex of
proteins to an overlapping sequence.

18. Nonsense mediated
A strong link between translation and RNA
turnover is also shown by nonsense-mediated
decay (NMD), which ensures that mRNAs
containing premature stop codons are degraded.
(evidence from yeaststudies)
NMD prevents the accumulation of aberrant
transcripts and truncated proteins by ensuring
rapid decay of the mRNAs.
Premature stop codons can target a
transcript for rapid, deadenylation-
independent decay.

19. ORF
5’
3’-UTR
stop
Normal stability
AAAAAAAA
NMD
ORF
5’
3’-UTR
stop
AAAAAAAA
Prematu
re stop

20. Conclusion
Controlling the rate at which the mRNA decays
can regulate the levels of cellular messenger RNA
transcripts. Because decay rates affect the
expression of specific genes, they provide a cell
with flexibility in effecting rapid change. mRNA
abundance is determined by balancing
transcription and RNA decay. mRNA stability can
be rapidly modulated to alter the expression of
specific genes thereby providing flexibility in
affecting changes in patterns of protein synthesis.

21. References
Molecular biology of the Gene: J.D. Watson
Molecular cell biology: Lodish 5
th
edition
Gene VIII: Lewin Benjamin
Google image