Poster abstracts

Poster number 95 submitted by Lankani Gunaratne

Understanding the function of the La protein in Trypanosoma brucei

G. Lankani Gunaratne (Ohio State Biochemistry Program, The Ohio State University), Ananth Casius (Department of Microbiology, The Ohio State University), Gabriel Silveira dAlmeida (Department of Microbiology, The Ohio State University), Richard J. Maraia (Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health), Juan D. Alfonzo (Department of Microbiology, The Ohio State University)

La is a ubiquitous nuclear RNA binding protein found in almost all eukaryotes investigated to date. Although this conservation suggests that La participates in an important process, its role in eukaryotic cells is not completely clear. Trypanosoma brucei, the causative agent of African Trypanosomiasis in humans, encodes a ‘classical’ genuine La protein (TbLa) with a La motif closely followed by an RNA recognition motif (RRM1), an additional RRM (RRM2α) and a C terminal short basic motif (SBM) along with a nuclear localization signal (NLS). We have shown that TbLa is essential for the survival of T. brucei through knocking down endogenous La by RNA interference (RNAi). A synonymous codon swapped version of myc-tagged TbLa, which is impervious to RNAi, (TbLa Rec) has been stably expressed in T. brucei and rescues a growth defect caused by RNAi down-regulation of endogenous La. Immunofluorescence microscopy showed that TbLa Rec resides mainly in the nucleus and partly in the cytoplasm, similar to endogenous La. Stable expression of an NLS-deleted TbLa Rec (TbLa NLSΔRec) in La RNAi cells rescues the growth phenotype similar to TbLa Rec. Immunofluorescence microscopy coupled with treatment of cells with Leptomycin B confirmed that TbLa with the NLS deletion indeed leads to exclusively a cytoplasmic localization of TbLa, but by rescuing the growth defect of endogenous La, it leads to the conclusion that the essential function of La is not necessarily due to its nuclear localization. Through polysome sedimentation analysis we show that polysome formation gets disrupted in the absence of TbLa which is rescued by TbLa Rec and TbLa NLSΔRec. This confirms TbLa plays an important role in ribosome biogenesis in T. brucei, a function TbLa is able to carry out while being limited to the cytoplasm. We have also constructed a series of myc-tagged recoded La mutants with each of the RRM, SBM and the NLS deleted. Through in vivo expression of individual RRM-deleted La mutants in endogenous La RNAi cells, we show that whereas RRM1 is essential for function, the second putative RRM2α and the SBM are dispensable. The latter raises questions about why RRM2α and SBM are maintained, other than for structural rather than functional reasons.

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Keywords: La protein, Trypanosoma brucei, RNA binding proteins