Abstract
CCCH zinc finger proteins (ZC3Hs) are a novel class of RNA-binding protein involved in post-transcriptional mechanisms controlling gene expression. We show TbZC3H20 from Trypanosoma brucei, the causative agent of sleeping sickness and other diseases, stabilizes two developmentally regulated transcripts encoding a mitochondrial carrier protein (MCP12) and trans-sialidase (TS-like E). TbZC3H20 is shown to be an RNA-binding protein that is enriched in insect procyclic form T. brucei and is the first ZC3H discovered controlling gene expression through modulating mRNA abundance in trypanosomes. Previous studies have demonstrated that RNA recognition motif-containing and PUF family RNA-binding proteins can control gene expression by stabilizing specific target mRNA levels. This work is the first to describe a ZC3H stabilizing rather than destabilizing target mRNAs as a regulatory mechanism and the first report of a ZC3H regulating a gene encoding a mitochondrial protein. This suggests a broader role for ZC3Hs in post-transcriptional regulation of gene expression than previously thought.
Highlights
Probes that are not Ͼ2 fold across all three arrays are shown in italics
To show the involvement of the ZC3H motifs within TbZC3H20 in binding directly to target transcripts, the third critical cysteine residue of both motifs were mutated to alanine, and recombinant TbZC3H20 CCCH zinc finger motif mutant (GST-20 ZFM) protein was used in an electrophoretic mobility shift assay with MCP12 and TS-like E 3Ј-UTR mRNAs (Fig. 5B)
PF cells treated with an inhibitor of protein synthesis revealed that two transcripts putatively regulated by TbZC3H20 (MCP12 and TS-like E) were stabilized in PF cells, and Northern analysis confirmed MCP12 and TS-like E are PF-enriched mRNAs
Summary
Kinetoplastids, such as Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major are protozoan parasites responsible for sleeping sickness, Chagas disease, and leishmaniasis in humans. Several studies have been conducted indicating developmental regulation of ϳ200 – 300 transcripts in each life cycle stage [12,13,14], and the differential abundance of enriched BF or PF transcripts are assumed to be governed via mRNA stability [13]. TbZFP3 has been investigated for a regulatory mechanism and found to control the expression of the variant forms of the insect stage-specific surface protein coat via translational repression rather than transcript stability [27]. The specific target mRNAs of TbZC3H20 defined in this study are important in the development of T. brucei, and understanding their regulation is a key part in understanding the life cycle of trypanosomes. This study is the first report of a ZC3H regulating a gene encoding a mitochondrial protein and the first to show ZC3Hs can regulate mRNA abundance (stability) as a post-transcriptional mechanism for regulating gene expression in trypanosomes. This work shows ZC3Hdependent stabilization rather than destabilization of a target mRNA, suggesting a broader involvement of ZC3Hs in regulating gene expression than previously thought
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