Cross-study analysis of genomic data defines the ciliate multigenic epiplasmin family: strategies for functional analysis in Paramecium tetraurelia

Abstract

Background: The sub-membranous skeleton of the ciliate Paramecium, the epiplasm, is composed of hundreds of epiplasmic scales centered on basal bodies, and presents a complex set of proteins, epiplasmins, which belong to a multigenic family. The repeated duplications observed in the P. tetraurelia genome present an interesting model of the organization and evolution of a multigenic family within a single cell.

Results: To study this multigenic family, we used phylogenetic, structural, and analytical transcriptional approaches. The phylogenetic method defines 5 groups of epiplasmins in the multigenic family. A refined analysis by Hydrophobic Cluster Analysis (HCA) identifies structural characteristics of 51 epiplasmins, defining five separate groups, and three classes. Depending on the sequential arrangement of their structural domains, the epiplasmins are defined as symmetric, asymmetric or atypical. The EST data aid in this classification, in the identification of putative regulating sequences such as TATA or CAAT boxes. When specific RNAi experiments were conducted using sequences from either symmetric or asymmetric classes, phenotypes were drastic. Local effects show either disrupted or ill-shaped epiplasmic scales. In either case, this results in aborted cell division. Using structural features, we show that 4 epiplasmins are also present in another ciliate, Tetrahymena thermophila. Their affiliation with the distinctive structural groups of Paramecium epiplasmins demonstrates an interspecific multigenic family.

Conclusion: The epiplasmin multigenic family illustrates the history of genomic duplication in Paramecium. This study provides a framework which can guide functional analysis of epiplasmins, the major components of the membrane skeleton in ciliates. We show that this set of proteins handles an important developmental information in Paramecium since maintenance of epiplasm organization is crucial for cell morphogenesis.

Figure 1

Comparative analysis of P. tetraurelia and T. thermophila epiplasmins using DNA sequence alignment. Five groups (1 to 5) were distinguished, based on node strength. According to hydrophobic cluster analysis, groups 1, 3 and 5 regroup 30 symmetric epiplasmins, group 2 represents 11 asymmetric epiplasmins, and group 4, 10 atypical epiplasmins. Results of large scale synteny analysis are reported as a red strand over the tree topology. Nodes corresponding to the 'ancient' and the 'old' WGD are circled in red and blue, respectively. EST numbers are compiled for each sub-group.

Figure 2

Modular organization of the P. tetraurelia epiplasmins, using generalized cluster analysis representation. For each protein, hydrophobic, loop and ambivalent clusters are represented by green, blue, and yellow blocks, respectively. Several structural domains, determined by HCA, are boxed in green for the central domain shared by the whole set, in yellow and pink for the hinge and Y rich domains, respectively.

Figure 3

Modular organization of the central domain of epiplasmins. A-Alignment of the seven repeated motifs in central domains of P. tetraurelia and T. thermophila epiplasmins. Two paralogs in P. tetraurelia and one T. thermophila protein share the first half of the epiplasmin central domain. B-HCA representation of these motifs is used to consider the position of conserved amino acids in relation with the hydrophobic clusters. This shows a spatial proximity between conserved proline and tyrosine (as mentioned in the discussion).

Figure 4

Evidence for common structural domains between P. tetraurelia epiplasmins (Epi) and T. thermophila epiplasmins (EpiT). HCA representation, where hydrophobic residues are shaded in yellow; helix breakers P, G, S, T in blue; alkaline residues in green and A, N, Q, D, E in pink.

Figure 5

5'UTR analysis of P. tetraurelia epiplasmin genes. The genes are regrouped according to EST numbers and presence of USE and TATA-like elements. These motifs are shaded in blue for TATA-like element, and in green or yellow for the USE motif according to whether it is located upstream or downstream from the TATA-like element, respectively.

Figure 6

Effects of RNA interference of genes coding for asymmetric and symmetric classes of epiplasmins on P. tetraurelia. A-Control Cells: a) Low magnification phase contrast and immunofluorescence with mAb CTS32 counterstained with DAPI. b) Higher magnification showing the alignment of cortical units along the kinetids. The arrow heads show normal pattern of scale duplication. B-Cells submitted to Epi 2 RNAi: a) Cell shape alteration at division time (boomerang) and 24 h later (plasmodial form). b) Irregularities in the alignment and the shape of the scales, and abnormally oriented striction of scales leading to 'double kinetids' (circled with white line) C-Cells submitted to Epi 41 RNAi: a) Same cell shape alteration as with Epi 2 at division time (boomerang) and 24 h later (plasmodial form). b) Numerous granules of epiplasmic material surround crenulated scales.