Endonuclease-mediated mRNA Decay Requires Tyrosine Phosphorylation of Polysomal Ribonuclease 1 (PMR1) for the Targeting and Degradation of Polyribosome-bound Substrate mRNA

Abstract

PMR1 is an endonuclease that is activated by estrogen to degrade Xenopus albumin mRNA. A previous report showed that the functional unit of endonuclease-mediated mRNA decay is a ϳ680-kDa polysome-bound complex that contains both PMR1 and substrate mRNA. PMR1 contains two domains involved in endonuclease targeting to polysomes, an N-terminal domain that lies between residues 200 and 250, and a C-terminal domain that lies within the last 100 residues. Loss of either domain inactivated PMR1 targeting to polysomes and stabilized albumin mRNA. The current study identified a phosphorylated tyrosine residue within the C-terminal polysome-targeting domain and showed that this modification is required for PMR1-mediated mRNA decay. Changing this tyrosine to phenylalanine inactivated the targeting of PMR1 to polysomes, blocked binding of PMR1 to the functional complex containing its substrate mRNA, prevented the targeting of a green fluorescent protein fusion protein to this complex, and stabilized albumin mRNA to degradation by PMR1 in vivo. A general tyrosine kinase inhibitor inhibited the phosphorylation of PMR1, which in turn inhibited PMR1-catalyzed degradation of albumin mRNA. These results indicate that one or more tyrosine kinases functions as a regulator of endonuclease-mediated mRNA decay.

Polysome-Bound Endonuclease PMR1 Is Targeted to Stress Granules via Stress-Specific Binding to TIA-1

“…Interestingly, a similar mechanism was recently reported for IRE1, which is activated by unfolded protein to cleave endoplasmic reticulumassociated mRNAs (16). For PMR1 to target polysomes and substrate mRNA, a tyrosine residue at position 650 within a consensus class I Src homology 2 sequence must be phosphorylated (37). Importantly, the overall process of endonucleasemediated mRNA decay depends on active translation of substrate mRNA (38), and inserting a stable stem-loop structure in the 5Ј untranslated region results in mRNA stabilization.…”

“…Bound complexes were eluted by cleavage with 50 U of Tev protease (Invitrogen) in 100 l of Tev buffer at 4°C for 2 h. Protein released from the resin was analyzed by Western blotting using the monoclonal antibody against the myc epitope tag for PMR1 and antibody to the FLAG epitope tag for TIA-1. To determine the impact of arsenite on tyrosine phosphorylation of PMR1, 2 ϫ 10 6 Cos-1 cells were transfected as described above with 10 g of myc-PMR60 o -TAP or GFP-TAP, and protein recovered following Tev protease cleavage of complexes bound to IgG-Sepharose was analyzed by Western blotting using antibodies to the myc epitope tag, anti-GFP, and the phosphotyrosine-specific monoclonal antibody PY20 as described previously (37). To determine the impact of arsenite-induced stress on the interaction of PMR1 with albumin mRNA, 2 ϫ 10 6 Cos-1 cells were transfected with 6 g of myc-PMR60 o -TAP or GFP-TAP plus 2 g of plasmid expressing albumin mRNA and 2 g of plasmid expressing firefly luciferase.…”

“…PMR60 is also present in a smaller complex (complex II) that sediments toward the top of sucrose gradients and at ϳ140 kDa on glycerol gradients. The latter is thought to be the precursor to complex I and the likely site where tyrosine phosphorylation occurs (37). The stressinduced phosphorylation of eIF-2␣ results in bulk dissociation of polysomes due to the block in reinitiation (22), although some mRNAs are resistant to this.…”

“…The binding of PMR60 o to complex I requires phosphorylation of the tyrosine residue at position 650 (37). To determine whether stress alters PRM60 o tyrosine phosphorylation, cytoplasmic extracts from control and arsenite-treated cells expressing myc-PMR60 o -TAP or GFP-TAP were absorbed with IgG-Sepharose, washed, and then eluted using Tev protease cleavage (Fig.…”

The poly(A)-limiting element enhances mRNA accumulation by increasing the efficiency of pre-mRNA 3′ processing

“…In the experiment in Figure 2 LM(tkÀ) cell lines expressing the tetracycline repressor were generated as described previously (Yang et al 2004) using a plasmid expressing the tetracycline repressor and a hygromycin resistance gene and clonal selection in hygromycin. These cells were transfected as above in medium lacking tetracycline, and reporter gene transcription was terminated at time 0 by adding 1 mg/mL of tetracycline to the medium.…”