summary of methods to assess mrna stability in eukaryotic cells
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Summary of methods to assess mRNA stability in eukaryotic cells. 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 - PowerPoint PPT PresentationTRANSCRIPT
Biologia molecolare - Robert F. Weaver Copyright © 2005 – The McGraw-Hill Companies srl
Summary of methods to assess mRNA stability in eukaryotic cellsSummary 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 sensitivityfor high abundance,
slow turnover mRNAs
Injection of in vitro transcribed 32P-
RNA
measurement of "true" chemical half life
lack of cellular RNA modifications, labour
intensive
oocytes can differ from somatic cells,
Inducible promoterrelatively rapid
inductioninduction may alter cell
physiologyHsp70 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 trans-acting 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 proteinsARE-binding proteinsProtein 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, GM-CSF, (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.
HuR
HuD
HuC
Hel-N1
TIAR 40, 42 RRM Brain, spleen, lung, liver,testis TNF, GM-CSF Transl. inhib.
TIA-1 Brain, spleen, testis
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.