Fig 1.
Analysis of mRNA populations associated with the three HA-tagged Leishmania infantum PABPs.
mRNA groups associated with the recombinant HA-tagged PABP1, PABP2 and PABP3 from L. infantum (Illumina sequencing). Enriched genes in at least two of three available RNA-seq datasets were manually classified and grouped using the gene ontology (GO) terms according to their molecular function. ‘Enriched’ means at least 2-fold more abundant than in the negative control.
Fig 2.
Protein-protein interaction assays investigating the direct binding between the L. infantum PABPs and their putative RBP23 and DRBD2 partners.
Pull down assays between radiolabeled RBP23 or DRBD2 and three recombinant PABPs. The upper panel shows the Coomassie-blue stained gel indicating the radiolabeled protein input, the GST recombinant (negative control) and the GST-PABP homologues. The panels below are autoradiographs showing the result of the interactions between radiolabeled RBP23 or DRBD2 and recombinant proteins. The RBP23 and DRBD2 recombinant proteins are indicated by arrows.
Fig 3.
Expression of HA-tagged RBP23 and DRBD2 proteins and the top- co-purifying partners with RBP23.
(A) Immunodetection of RBP23-HA or DRBD2-HA with monoclonal anti-HA antibody in whole L. infantum cell extracts expressing RBP23-HA or DRBD2-HA. (B) Immunodetection of the two HA-tagged proteins after immunoprecipitations with anti-HA beads using cytoplasmic extracts from the same L. infantum lines as well as from cells lacking HA-tagged proteins (IP control). The red arrows highlight the immunoprecipitated proteins, with the asterisks indicating IgG related bands. (C) Bar chart with proteins co-precipitated with RBP23 and having an enrichment strength defined as ‘+++’ (orange) and ‘++’ (green), according to Table 2. Proteins co-precipitated with both L. infantum HA-tagged PABP1 [33] and RBP23 are indicated by arrows. Proteins found to co-precipitate also with DRBD2 are underlined.
Table 1.
Proteins specifically co-immunoprecipitating with L. infantum RBP23.
Fig 4.
Top proteins co-purifying with HA-tagged DRBD2 and their subcellular compartmental localization compared to HA-tagged RBP23.
(A) Bar chart with proteins co-precipitated with DRBD2 with the enrichment strength defined as ‘+++’ (orange), ‘++’ (green) and ‘+’ (blue), according to Table 2. Proteins co-purifying with both T. cruzi DRBD2 [45] and its L. infantum HA-tagged orthologue are indicated by arrows. Proteins in common with RBP23 are underlined. (B) Pie charts with subcellular localization of DRBD2 (left chart) or RBP23 (right chart) partners. This was defined based on cellular component classes: cytoskeleton; cytoplasm; mitochondrion; nuclear; organelles/granules (except mitochondria); n/d or not defined. For uncharacterized proteins, their localization was defined based on their respective orthologues from T. brucei and according to TrypTag.org.
Table 2.
Proteins specifically co-immunoprecipitating with L. infantum DRBD2.
Fig 5.
Comparative enrichment ratios for the major protein groups co-precipitated with L. infantum HA-tagged RBP23 and DRBD2.
Proteins co-precipitated with RBP23 and DRBD2 belonging to the selected functional categories related to mRNA processes are represented by boxes coloured according to the enrichment ratios derived from S3 and S5 Tables. The numbers inside the boxes indicate the number of subunits of the named complex (including ribosomes) that have equivalent enrichment ratios. (A) Enrichment ratios for the baits and three PABP homologues. (B) Enrichment ratios for eIF4F subunits and eIF4F interacting proteins. (C) Enrichment ratios for other proteins with functions related to mRNA splicing, polyadenylation, translation or degradation.
Fig 6.
Analysis of mRNA populations associated with the HA-tagged RBP23 and DRBD2.
(A) mRNA groups associated with RBP23. (B) mRNA groups associated with DRBD2. Transcripts were classified and grouped as detailed in Fig 1, with an extra category added to specifically highlight the mitochondrial proteins.
Fig 7.
Sequence elements within the 3’UTRs of trypanosomatid mRNAs associated with RBP23 and DRBD2.
(A) Comparison of RBP23 (blue) and DRBD2 (orange) 5’ (▼) and 3’ (▲) UTRs, CDS (■) and total transcript lengths (●) according to L. donovani orthologous sequences [48]. The mean values are represented by black lines and the RBP23 transcript is indicated by the same symbols coloured in black. (B) Putative motifs found within 3’UTRs from the top 20 mRNAs associated to either HA-tagged RBP23 or DRBD2 in L. infantum and L. donovani. The motifs found in searches with mRNAs enriched with the HA-tagged RBP23, including the RBP23 transcript as well as the top 19 mRNAs encoding ribosomal proteins, are shown on the left. Those motifs found for the top 20 mRNAs associated to HA-tagged DRBD2 are shown on the right. All logos were generated by MEME (Multiple Em for Motif Elicitation) and the relative height of each nucleotide represents the conservation at that specific position. The e-values are in the right corner of each logo.
Fig 8.
Proposed role for RBP23 in mediating the association of mRNAs encoding ribosomal proteins with PABP1 and the EIF4E4/EIF4G3/EIF4AI complex.
According to the proposed model, RBP23 recognizes target mRNAs through motifs within their 3’UTRs. It might recognize these mRNAs on its own or already bound to PABP1. PABP1 then mediates the recruitment of the translation initiation complex EIF4E4/EIF4G3/EIF4AI. It is also possible that PABP1 might already be bound to the complex prior to mRNA recognition but this is not shown. Both PABP1 and EIF4E4 are also phosphorylated during exponential growth, allowing for a more refined regulation of their activity and, presumably, the translation of the ribosomal protein mRNAs.