Characterization of deadenylation in trypanosome extracts and its inhibition by poly(A)-binding protein Pab1p

Abstract

The stability of mRNAs is an important point in the regulation of gene expression in eukaryotes. The mRNA turnover pathways have been identified in yeast and mammals. However, mRNA turnover pathways in trypanosomes have not been widely studied. Deadenylation is the first step in the major mRNA turnover pathways of yeast and mammals. To better understand mRNA degradation processes in these organisms, we have developed an in vitro mRNA turnover system that is functional for deadenylation. In this system, addition of poly(A) homopolymer activates the deadenylation of poly(A) tails. The trypanosomal deadenylase activity is a 3'-->5' exonuclease specific for adenylate residues, generates 5'-AMP as a product, is magnesium dependent, and is inhibited by neomycin B sulfate. These characteristics suggest similarity with other eukaryotic deadenylases. Furthermore, this activity is cap independent, indicating a potential difference between the trypanosomal activity and PARN, but suggesting similarity to Ccr4p/Pop2p activities. Extracts immunodepleted of Pab1p required the addition of poly(A) competition to activate deadenylation. Trypanosomal Pab1p functions as an inhibitor of the activity under in vitro conditions. Pab1p appears to be one of several mRNA stability proteins in trypanosomal extracts.

FIGURE 1.

Addition of poly(A) homopolymer to trypanosomal cytoplasmic extracts activates a distributive nuclease activity. (A) Internally radiolabeled MCS A60 RNA (124 nt) substrate was incubated for 30 min in L. seymouri cytoplasmic extract. (B) Internally radiolabeled SL-MCS ERA A60 RNA (209 nt) was incubated with decreasing amounts of L. seymouri extracts for 60 min as indicated. A total of 500 ng poly(A) homopolymer was added to each reaction. (C) Internally radiolabeled Gem A60 RNA (125 nt) was incubated for 60 min in T. brucei cytoplasmic extract. Increasing amounts of poly(A) or poly(C) were added to the reactions in A and B as indicated. Reaction products were analyzed on a 7-M urea 5% polyacrylamide gel following phenol extraction and ethanol precipitation. The A60 arrow indicates the input RNA substrate and the A0 arrow indicates the deadenylated form of the RNA substrate; MCS A0 (64 nt), SL-MCS ERA A0 (149 nt), and Gem A0 (65 nt), respectively, in A, B, and C. The lanes marked I indicate the input RNA. The RNA A0 marker is shown in lanes marked M. An additional RNA marker (165 nt) was added to lane M in B.

FIGURE 2.

Poly(A)-activated nuclease activity is a 3′→5′ deadenylase with specificity for adenylate residues. (A, left panel) Reaction products generated in a time-course incubation of two different internally radiolabeled RNA substrates in L. seymouri extracts. The SL-MCS ARE A60 (209 nt) and SL-MCS ARE A60+20 (229 nt) substrates consist of plasmid polylinker sequence followed by a 60-nt poly(A) tail or a 60-nt poly(A) tail, followed by 20 additional non-poly(A) nucleotide at the 3′ end. (A, right panel) A similar experiment, except that Gem A60 (125 nt) and Gem A60+20 (145 nt) RNAs were incubated with T. brucei cytoplasmic extracts. (B,C) Parallel time courses were performed using L. seymouri or T. brucei extracts with RNA substrates possessing a 60-nt poly(A) tail or a poly(A) tail interrupted by a 9-nt poly(C) tract. SL-MCS A60 (169 nt) and SL-MCS A30 C(9) A30 (178 nt) was used with L. seymouri extract, and Gem A60 (125 nt) and Gem A30 C(9) A30 (134 nt) was used with T. brucei extract. The migration of decay intermediates after incubation are shown. Below each data set, these data are represented graphically as the distance of migration of the RNA substrate in reference to the RNA A0 marker. The graphs were generated by a continuity plot analysis of each lane, and the peak of the signal intensity was set as the point that represented distance of migration. (▪) Uninterrupted RNA substrate decay; (□) decay of the interrupted poly(A) tail RNA substrate. All reaction products were separated using 7-M urea 5% polyacrylamide gels following phenol extraction and ethanol precipitation. The A60 arrow indicates the input RNA with a poly(A) tail of 60 nt, and the A0 arrow indicates the deadenylated form of the RNA substrate. The lanes marked I indicate the input RNA. The SL-MCS ARE A0 (149 nt), SL-MCS A0 (109 nt), and Gem A0 RNA A0 (65 nt) markers were resolved in lanes marked M (A–C).

FIGURE 3.

The trypanosomal deadenylation activity has characteristics similar to other known deadenylases. (A) Parallel titrations of neomycin B sulfate were performed with L. seymouri, T. brucei, and HeLa cell extracts. Deadenylation activity was assessed as described above. (B) The divalent cation requirement of the trypanosomal deadenylase activity was determined by inclusion of 10 mM EDTA in the reaction. Reaction products were analyzed by separation on 7-M urea 5% polyacrylamide gels following phenol extraction and ethanol precipitation. Arrows indicate the adenylated substrate (A60) and the deadenylated substrate (A0). Input RNA is in the lane labeled I. The RNA A0 marker is in the lane labeled M. (C) One-dimensional TLC analysis of the deadenylation reaction products. RNAs radiolabeled at the poly(A) tail were incubated in trypanosomal extracts for 60 min. The reaction products were phenol-chloroform extracted and resolved using PEI-cellulose TLC plates. The positions of UV-shadowed markers corresponding to 5′-AMP and 3′-AMP are indicated.

FIGURE 4.

The trypanosomal deadenylase activities are cap independent and unaffected by AU-rich elements in vitro. (A, left panel) Internally radiolabeled SL-MCS ARE A60 (209 nt) RNA possessing a triphosphate cap incubated in L. seymouri cytoplasmic extracts for the indicated times. (Right panel) A parallel time course in L. seymouri extracts using [γ-³²P]-7-methyl-guanosine cap-labeled SL-MCS ARE A60 RNA (209 nt). (B) Internally radiolabeled polyadenylated Gem A60 RNA (125 nt) containing either a m⁷G cap or a 5′ triphosphate end were incubated in T. brucei extracts. (C) Internally labeled RNAs, SL-MCS ARE A60 (209 nt) and SL-MCS ERA A60 (209 nt), containing a 60-nt poly(A) tail, and either an ARE element or the reverse sequence (ERA) were incubated in L. seymouri extracts. Adjacent to the gel image is a graphical representation of the data as described in Figure 2 ▶. (□) Decay of the ARE containing RNA; (▪) Decay of the ERA containing RNA. Reaction products were separated using 7-M urea 5% polyacrylamide denaturing gels following phenol extraction and ethanol precipitation. The arrows indicate the adenylated form (A60) and deadenylated form (A0) of the RNA substrate. The lanes labeled I are input RNA. The lanes labeled M are SL-MCS ARE A0 RNA (149 nt) and Gem A0 (65 nt).

FIGURE 5.

Pab1p is not the only inhibitor of deadenylation in trypanosomal extracts. (A) Western blot analysis of L. seymouri cytoplasmic extract (Ls ext.) and purified recombinant L. major Pab1p (LmPab1p) using a rabbit polyclonal antibody to L. major Pab1p is shown. (B) Immunoprecipitation of Pab1p from L. seymouri extracts was determined by analysis of the depleted extract using Western blots. Four rounds of immunoprecipitation were performed using LmPab1 antisera or rabbit normal sera. Cytoplasmic extract (CE) and precleared cytoplasmic extracts (PC) are indicated. Binding of the Pab1p to the antibody-bead complex was confirmed by Western blotting (data not shown). (C) Fourth-round Pab1p and normal sera-depleted extracts were tested for deadenylation activity using SL-MCS A60 (169 nt) RNA substrate. Parallel 60-min reactions using either treated extract are shown with or without the addition of poly(A) homopolymer. The lanes labeled M indicate SL-MCS A0 RNA (109 nt) as a marker.

FIGURE 6.

Pab1p is an inhibitor of deadenylation in trypanosome extracts. (A) L. seymouri cytoplasmic extracts were incubated batch-wise with poly(A) Sepharose or Protein G Sepharose resin. The deadenylation activity of the supernatants were determined using Gem A60 RNA (125 nt) substrate with and without the addition of poly(A) homopolymer. (Left panel) The deadenylation activity of the treated extract from the poly(A) Sepharose incubation; (right panel) the deadenylase activity of the Protein G Sepharose-treated extract. (B) The poly(A)-independent extract was incubated with ~100 ng of BSA (left panel) or purified recombinant LmPab1p (right panel). Reaction products were separated using 7-M urea 5% polyacrylamide gels. Arrows indicate the polyadenylated RNA substrate (A60) and the deadenylated substrate (A0) Gem A0 (65 nt).

FIGURE 7.

Deadenylation is triggered by removal of RNA-binding proteins that interact with the poly(A) tail. (1) mRNA is normally protected from deadenylation by RNA-binding proteins, including Pab1p. (2) Addition of poly(A) homopolymer to extracts results in removal of poly(A)-binding proteins, including Pab1p, allowing deadenylation of the poly(A) tail. (3) Addition of Pab1p to unprotected poly(A) tails prevents deadenylation. (4) Removal of Pab1p from extracts does not abrogate the requirement for poly(A) homopolymer addition to activate deadenylation.