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. 2000 Mar 1;28(5):1211-20.
doi: 10.1093/nar/28.5.1211.

Poly(A)-binding protein I of Leishmania: functional analysis and localisation in trypanosomatid parasites

E J Bates  1 , E Knuepfer, D F Smith
Affiliations

Poly(A)-binding protein I of Leishmania: functional analysis and localisation in trypanosomatid parasites

E J Bates et al. Nucleic Acids Res. .

Abstract

Regulation of gene expression in trypanosomatid parasites is predominantly post-transcriptional. Primary transcripts are trans-spliced and polyadenylated to generate mature mRNAs and transcript stability is a major factor controlling stage-specific gene expression. Degenerate PCR has been used to clone the gene encoding the Leishmania homologue of poly(A)-binding protein (Lm PAB1), as an approach to the identification of trans-acting factors involved in this atypical mode of eukaryotic gene expression. lmpab1 is a single copy gene encoding a 63 kDa protein which shares major structural features but only 35-40% amino acid identity with other PAB1 sequences, including those of other trypanosomatids. Lm PAB1 is expressed at constant levels during parasite differentiation and is phosphorylated in vivo. It is localised predominantly in the cytoplasm but inhibition of transcription with actinomycin D also reveals diffuse localisation in the nucleus. Lm PAB1 binds poly(A) with high specificity and affinity but fails to complement a null mutation in Saccharomyces cerevisiae. These properties are indicative of functional divergence in vivo.

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Figures

Figure 1
Figure 1
Alignment of LmPAB1 with PAB1 homologues. The open reading frame of LmPAB1 is aligned (CLUSTALV; 61) against PAB1 homologues from Human (H.s) [genpept accession no. g129617], S.cerevisae (S.c) [genpept accession no. P04147] and T.cruzi (T.c) [GenBank accession no. U06070]. Coloured boxes delineate the four N-terminal RNA binding domains (RBD1, blue, amino acids 22–105; RBD2, red, amino acids 106–193; RBD3, green, amino acids 194–288; RBD4, orange, amino acids 303–386) and the conserved C-terminal domain (purple, amino acids 498–560). RNP1 motifs are highlighted (asterisks) and potential sites of phosphorylation indicated (circles) (35).
Figure 2
Figure 2
Southern analysis of L.major genomic DNA. (A) Partial restriction map of the lmpab1 locus. Regions encoding the RNA binding domains and the C-terminal domain are boxed and highlighted differentially. Positions of the initiator and stop codons and extent of probes B and C are shown. Internal restriction sites are indicated: N, NotI; P, PstI; X, XhoI. (B) Southern hybridisation of probes B and C to L.major genomic DNA digested with XhoI (lanes 1 and 6); PstI (lanes 2 and 7); NotI (lanes 3 and 8); EcoRI (lanes 4 and 9); ClaI (lanes 5 and 10). Blots were hybridised and washed at low stringency, as described.
Figure 3
Figure 3
Analysis of the expression pattern of LmPAB1. (A) Total RNA, isolated from differentiating parasites over a time course (3–8 days), was size-separated, blotted and probed with the open reading frame of lmpab1. The same blot was re-probed with RPS8 (constitutively expressed, S8 ribosomal protein gene) and β-tubulin (which recognises differentially expressed transcripts of 2.2 and 3.2 kb; 45). (B) Expression of the lmpab1 transcript normalised against RPS8 expression and plotted as a ratio of day 3 (logarithmic parasite) expression levels. (C) Expression of LmPAB1 in differentiating parasites. Total proteins were extracted at daily intervals (3–7 days), separated by 10% SDS–PAGE, electroblotted and identical blots probed with abSK375 (anti-LmPAB1, top panel) and ab366 (anti-HASPB, middle panel). Silver staining was used to visualise protein loading (bottom panel). (D) In vivo phosphorylation of LmPAB1. Total lysate from 32P-radiolabelled parasites was immunoprecipitated (IP tracks) with anti-LmPAB1 or pre-immune serum, prior to analysis by 8% SDS–PAGE. The IP/LmPAB1 track was then immunoblotted against anti-LmPAB1 (I track). Relative exposure times at –70°C: total, 1 × 107 parasites labelled with 100 µCi 32P, 14 h; IP, 1 × 108 parasites labelled with 200 µCi 32P, 96 h. (E) Cross-reactivity of anti-LmPAB1with PAB1 from other species. Cell extracts from L.major (Lm, 5.0 × 106), T.brucei procyclics (Tb, 2.0 × 107), S.cerevisiae (Sc, 2.0 × 107) and mouse macrophages (Mm, 2.0 × 106) were separated by 10% SDS–PAGE and immunoblotted with anti-LmPAB1.
Figure 4
Figure 4
RNA binding analysis. (A) Binding of cytoplasmic parasite proteins to end-labelled poly(A). Reactions were performed at low (0.15 M) or high salt (1 M NaCl); unlabelled poly(A) competitor was added as indicated to reactions containing low salt. Complexes were UV cross-linked and separated by 10% SDS–PAGE followed by autoradiography. N, no lysate added. (B) Immunoprecipitation of LmPAB1:poly(A) complexes bound at low (0.15 M) or high (1 M) salt, with anti-LmPAB1 or with pre-immune serum (Pre), followed by 10% SDS–PAGE separation and autoradiography (4 days at –70°C).
Figure 5
Figure 5
Transcriptional inhibition affects the cellular localisation of LmPAB1. (A) Immunofluorescent detection of LmPAB1 (with abSK375; IF) and DAPI staining of nuclear (N) and kinetoplast (K) DNA in late logarithmic phase parasites, untreated (–) or incubated with 10 µg/ml actinomycin D (+) for 10 h prior to fixing and permeabilisation. Scale bar = 5 µm. (B) Parasite fractions were separated (in the presence of protease inhibitors) and immunoblotted with anti-LmPAB1 (8% SDS–PAGE) or ab415 (Sw3, anti-histone H1; 10% SDS–PAGE). Lane 1, total lysate, 5 × 106 cells; lane 2, cytoplasmic fraction, 5 × 106 cells; lane 3, nuclear fraction, 5 × 106 cells; lane 4, nuclear fraction, 3 × 107 cells. (C) Parasites treated with 0–20 µg/ml of actinomycin D for 10 h were fractionated (in the absence of protease inhibitors) and analysed by 6% SDS–PAGE and immunoblotting with anti-LmPAB1. Chemiluminescent exposures were for 15 s [total (T) and cytoplasmic (C) fractions from 2.5 × 107 cells] or 1 min [nuclear fraction (N) from 4.5 × 107 cells]. (D) Cytoplasmic fractions from 2 × 106 parasites incubated with actinomycin D for 24 h at 26°C or 4°C as indicated. LmPAB1 was immunodetected by chemiluminescence, exposure time 8 min.
Figure 5
Figure 5
Transcriptional inhibition affects the cellular localisation of LmPAB1. (A) Immunofluorescent detection of LmPAB1 (with abSK375; IF) and DAPI staining of nuclear (N) and kinetoplast (K) DNA in late logarithmic phase parasites, untreated (–) or incubated with 10 µg/ml actinomycin D (+) for 10 h prior to fixing and permeabilisation. Scale bar = 5 µm. (B) Parasite fractions were separated (in the presence of protease inhibitors) and immunoblotted with anti-LmPAB1 (8% SDS–PAGE) or ab415 (Sw3, anti-histone H1; 10% SDS–PAGE). Lane 1, total lysate, 5 × 106 cells; lane 2, cytoplasmic fraction, 5 × 106 cells; lane 3, nuclear fraction, 5 × 106 cells; lane 4, nuclear fraction, 3 × 107 cells. (C) Parasites treated with 0–20 µg/ml of actinomycin D for 10 h were fractionated (in the absence of protease inhibitors) and analysed by 6% SDS–PAGE and immunoblotting with anti-LmPAB1. Chemiluminescent exposures were for 15 s [total (T) and cytoplasmic (C) fractions from 2.5 × 107 cells] or 1 min [nuclear fraction (N) from 4.5 × 107 cells]. (D) Cytoplasmic fractions from 2 × 106 parasites incubated with actinomycin D for 24 h at 26°C or 4°C as indicated. LmPAB1 was immunodetected by chemiluminescence, exposure time 8 min.
Figure 6
Figure 6
Functional complementation in S.cerevisiae. (A) Galactose inducible expression of LmPAB1 in yeast strain YAS2031. YAS2031 (pAS77) transformed with empty vector p415 or with vector carrying lmpab1 (p415A) and an untransformed control (n/a) were grown in the presence (+) or absence (–) of galactose. Expression of LmPAB1 was detected by immunoblotting and chemiluminescence. Lm, L.major total cell lysate (1 × 107 cells). (B) Yeast strain YAS2031 and control strain YDM146, transformed with empty vector (p416/p415) or vector plus lmpab1 (p416A/p415A) plated on to selective media including 5′-fluoro-orotic acid and galactose, as the sole carbon source.
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