Transcript Summary of methods to assess mRNA stability in eukaryotic cells

Biologia molecolare - Robert F. Weaver
Copyright © 2005 – The McGraw-Hill Companies srl
Summary of methods to assess mRNA stability in eukaryotic cells
Method
Advantage
Disadvantage
Comments
Pulse-chase
labeling with 3H-U
measurement of "true"
chemical half life
low sensitivity
for high abundance,
slow turnover mRNAs
Injection of in vitro
transcribed 32PRNA
measurement of "true"
chemical half life
lack of cellular RNA
modifications, labour
intensive
oocytes can differ
from somatic cells,
Inducible promoter
relatively rapid
induction
induction may alter cell
physiology
Hsp70 and myc
promoter
Pharmacological
transcription block
can be applied to all
genes, rapid onset
block
perturbation of cellular
metabolism,
Actinomycin D, and
DRB most commonly
used
Comparison of
transcription rate
and steady state
mRNA level
can be applied to all
genes, useful
screening procedure
mRNA stability is not
directly measured
should only be used
in combination with
another method
In vitro RNA
degradation
easy, identification of
intermediates,
purification of transacting factors
difficult to establish
physiological
relevance and
specificity must be
established
mRNA degradative activities in mammalian cells
Decapping
• DCP2 which binds RNA as a prerequisite for cap recognition.
• DCP1 augments DCP2 activity
• LSM (SM-LIKE) PROTEINS augment DCP2 activity
5’ -to-3’ exonuclease activity
• XRN1 is a proven 5’ -to-3’ exonuclease that localizes to the cytoplasm.
• RAT1/XRN2 is only thought to be a 5’ -to-3’ exonuclease on the basis of its similarity to
the yeast orthologue.
Deadenylation
• PARN is one of five mammalian homologues to yeast Caf1/Pop2 protein
3’ -to-5’ exonuclease activity
• Exosome (six RNase-PH-DOMAIN components, PM/SCL75,MTR3,RRP41, RRP42,
RRP43 and RRP46; three S1 and KH RNA-binding components,RRP4, RRP40 and
CSL4; the RNASE D-like components PM/SCL100; the putative helicaseKIAA0053; and
a protein that is phosphorylated in the M phase of the cell)
PMR1-like activity
• Polysomal ribonuclease 1 (PMR1) is a polysome-associated mRNA endonuclease
ARE-binding proteins
Protein
kDa
Motif
Expression site
ARE
Function
AUF1
37,40,42,45
RRM
Ubiquitous
c-myc, c-fos, GM-CSF
mRNA destab.
AUBF
ND
ND
T cells
c-fos, INF, IL-3 v-myc, GMCSF, (AUUUA)n
ARE-binding
corr. with mRNA
stab.
AU-A
34
ND
T cells
TNF, GM-CSF, c-myc
ND
AU-B
30
AU-C
43
hnRNPA1
36
RRM
Human PBMCs
GM-CSF, IL-2, c-myc
ND
hnRNPC
43
Elav-like
36–40
RRM
Ubiquitous, nervous system
c-myc, c-fos,TNF-a,GM-CSF
mRNA stab.
40, 42
RRM
Brain, spleen, lung, liver,testis
TNF, GM-CSF
Transl. inhib.
HuR
HuD
HuC
Hel-N1
TIAR
Brain, spleen, testis
TIA-1
TTP
44
Cys3His
Fibroblasts, macrophages
TNF, IL-3 GM-CSF
mRNA destab.
KSRP
78
KH
Neural cells and other types
c-fos
mRNA destab.
•AUBF, AU binding factor ; AU-A, AU binding factor-A ; AU-B, AU binding factor-B ; AU-C, AU binding factor-C ; hnRNP,
heterogeneous nuclear ribonucleoprotein ; KH, hnRNP-K homology domain; KSRP, KH-type splicing regulatory protein 1; ND,
not determined; PBMC, peripheral blood mononuclear cell.