2. Biologia molecolare - Robert F. Weaver Copyright © 2005 – The McGraw-Hill Companies srl

5. Summary of methods to assess mRNA stability in eukaryotic cells MethodAdvantageDisadvantageComments 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 32P- RNA 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 trans- acting factors difficult to establish physiological relevance and specificity must be established

7. 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

10. ARE-binding proteins ProteinkDa MotifExpression siteAREFunction AUF1 37,40,42,45RRMUbiquitousc-myc, c-fos, GM-CSFmRNA destab. AUBF ND T cellsc-fos, INF, IL-3 v-myc, GM- CSF, (AUUUA)n ARE-binding corr. with mRNA stab. AU-A 34NDT cellsTNF, GM-CSF, c-mycND AU-B 30 AU-C 43 hnRNPA1 36RRMHuman PBMCsGM-CSF, IL-2, c-mycND hnRNPC 43 Elav-like 36–40RRMUbiquitous, nervous systemc-myc, c-fos,TNF-a,GM-CSFmRNA stab. HuR HuD HuC Hel-N1 TIAR 40, 42RRMBrain, spleen, lung, liver,testisTNF, GM-CSFTransl. inhib. TIA-1 Brain, spleen, testis TTP 44Cys3HisFibroblasts, macrophagesTNF, IL-3 GM-CSFmRNA destab. KSRP 78KHNeural cells and other typesc-fosmRNA 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.