Abstract
Leishmania parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few Leishmania trans-regulators are known. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main Leishmania lifecycle stages. Supporting the validity, although the crosslinked RBPome is magnitudes more enriched, the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in L. mexicana parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression versus RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the trans-regulatory mRNA:Protein (mRNP) complexes that drive Leishmania parasite lifecycle progression.
Highlights
Decisive evidence that trans-regulator RNA binding proteins (RBPs) can selectively bind distinct mRNA target pools in a stage-regulated manner despite equivalent transcript levels
Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins and whole cell proteomes from the three main Leishmania lifecycle stages
Isolation and Validation of L. mexicana Life Cycle Stages— For the study of the different mRNA binding proteomes during the L. mexicana lifecycle, 4 biological samples corresponding to the 3 main lifecycle stages were isolated and molecularly verified
Summary
A comprehensive, quantified identification of the mRNAbinding and whole cell proteomes in the three main Leishmania lifecycle stages, the first such comparison in kinetoplastid parasites, demonstrates transregulator RBPs select distinct, specific mRNA target pools in a stage-regulated manner despite equivalent, constitutive transcript levels available. Leishmania spp. parasites are the causative agent of leishmaniasis, a neglected disease that represents the ninth largest global infectious disease burden [1] These protozoa have a dixenous lifecycle that transitions between multiple promastigote stages in the sandfly vector to the amastigote stage in the phagolysosomes of mammalian immune cells [2]. The Leishmania mexicana RBPome Is Stage Regulated were refined in scope to only one lifecycle stage, yet importantly confirm that mRNA-bound factors in kinetoplastid cells include proteins without canonical RNA-binding motifs (14 –18). These findings may implicate post-translational modifications in the fluctuations of RNA binding potential and relative stability of candidate transregulators as well as overall cellular translational capacity and activity
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