Abstract
Trypanosoma brucei is the causative agent of human sleeping sickness. The parasites' variant surface glycoprotein (VSG) enables them to evade adaptive immunity via antigenic variation. VSG comprises 10% of total cell protein and the high stability of VSG mRNA is essential for trypanosome survival. To determine how VSG mRNA stability is maintained, we used mRNA affinity purification to identify all its associated proteins. CFB2 (cyclin F-box protein 2), an unconventional RNA-binding protein with an F-box domain, was specifically enriched with VSG mRNA. We demonstrate that CFB2 is essential for VSG mRNA stability, describe cis acting elements within the VSG 3'-untranslated region that regulate the interaction, identify trans-acting factors that are present in the VSG messenger ribonucleoprotein particle, and mechanistically explain how CFB2 stabilizes the mRNA of this key pathogenicity factor. Beyond T. brucei, the mRNP purification approach has the potential to supply detailed biological insight into metabolism of relatively abundant mRNAs in any eukaryote.
Highlights
In the cytosol, eukaryotic mRNAs interact with numerous proteins that influence mRNA folding, localization, translation efficiency and longevity (1)
We demonstrate that CFB2 is essential for Variant Surface Glycoprotein (VSG) mRNA stabil18 ity, describe cis acting elements within the VSG 3'-untranslated region that regulate the 19 interaction, identify trans-acting factors that are present in the VSG messenger ribonu20 cleoprotein particle and mechanistically explain how CFB2 stabilizes the mRNA of this 21 key pathogenicity factor
Antigenic variation is effected through a combination of expression site transcription switching, and genetic rearrangements that replace the currently expressed VSG gene with a different one from a repertoire of alternative VSG genes located at telomeres or in sub-telomeric arrays (1369 15)
Summary
Eukaryotic mRNAs interact with numerous proteins that influence mRNA folding, localization, translation efficiency and longevity (1). 47 The genome organization of trypanosomes and related organisms is unusual, since protein coding genes lack individual promoters. Instead, they are arranged in long polycistronic transcription units that are constitutively transcribed by RNA polymerase II. Association of the active VSG expression site with the site of spliced leader RNA production results in extremely efficient processing of the VSG mRNA (16). These measures alone do not suffice: the VSG mRNA is extremely
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