Analysis of mRNA deadenylation by multi-protein complexes

Abstract

Graphical abstract

Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo

“…To demonstrate that the lower band shown in Fig. 1D, lanes 3 and 4, was the deadenylated species of the target transcript, we removed the poly(A) tails from mRNA with oligo(dT) [12][13][14][15][16][17][18] plus RNase H, as described previously (22) (Fig. 1E).…”

“…Full-length mouse TTP partially complements a deficiency in the TTP family member Zfs1 in S. pombe (24). Therefore, we examined the activities of recombinant WT and mutant forms of human TTP in a deadenylation assay involving purified, recombinant CCR4-NOT complexes from S. pombe that were expressed in and purified from insect cells (14,15). Full-length, purified recombinant WT TTP, in the form of an N-terminal fusion with maltose binding protein (MBP), stimulated the deadenylation activity of the CCR4-NOT complex when tested against a synthetic mRNA target that contained a single ARE ( Fig.…”

“…Briefly, total cellular RNA samples from cells (without ActD treatment) cotransfected with Mlp-TNF3= and various TTP or GFP expression constructs were incubated at 55°C in 50 mM KCl with or without 0.6 g of oligo(dT) 12-18 for 5 min, followed by another 20 min at 25°C. All reaction mixtures included 10 g of RNA, except for the GFP and C124R mutant samples incubated with RNase H, which included 5 g. Reaction buffer containing RNase H (1 U per reaction sample) was added to samples annealed with oligo(dT) [12][13][14][15][16][17][18] , or reaction buffer without the enzyme was added to samples without oligo(dT) [12][13][14][15][16][17][18] , followed by incubation at 37°C for 1 h. After precipitating the RNA, the samples were used for Northern blotting as described above.…”

“…Cell-free deadenylation assays were performed as described previously (15,25), using a target mRNA sequence at a final concentration of 200 nM that contained a single TTP family member binding site, 5=-[6FAM/fluorescein]-AAUCAUCCUUAUUUAUUACCAUUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3= (the binding site is underlined). Recombinant purified CCR4-NOT complexes from S. pombe were incubated in the presence or absence of identical concentrations (60 nM) of the following recombinant MBP fusion proteins: wild-type full-length human TTP, its C-terminally truncated mutant (ΔCNDB), its nonbinding C118R zinc finger mutant, its TZF domain alone, or the MBP protein alone.…”

“…We addressed this in two mouse genetic backgrounds, C57BL/6 (B6) and 129, in both sexes, and in both mono-and diallelic comparisons. We also investigated the ability of recombinant full-length and C-terminally truncated TTP to promote deadenylation of target mRNAs by the recombinant CCR4-NOT complex from Schizosaccharomyces pombe (14,15) and to substitute for the TTP family member Zfs1 in living S. pombe cells (16,17).…”