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The Leishmania tarentolae spliced leader contains determinants for association with polysomes
Gusti M Zeiner1, Nancy R Sturm, David A Campbell
1Department of Microbiology, Immunology, and Molecular Genetics, University of California at Los Angeles, Los Angeles, California 90095-1489, USA.
Abstract:
In kinetoplastids, every nuclear-derived mRNA contains an identical 39-nucleotide (nt) spliced leader at its 5'-terminus. The spliced leader is derived from substrate spliced leader RNA and joined to pre-mRNA by trans-splicing, thus providing mature mRNAs with an m7G cap and additional methylations referred to as cap 4. It was shown previously that mutations spanning nucleotides 10-39 of the spliced leader did not affect substrate spliced leader RNA transcription or trans-splicing in Leishmania tarentolae (Saito, R. M., Elgort, M. G., and Campbell, D. A. (1994) EMBO J. 13, 5460-5469). In this study we examined these sequences for a possible role in translation by assaying the association of mRNAs, which possess mutated spliced leaders, with polysomes. For the nt 28-39 mutated spliced leaders, both the substrate spliced leader RNA and the spliced leader demonstrated a wild-type methylation pattern; spliced nt 28-39 mRNA was found in polysomes. Thus, the nt 28-39 region conserved primary sequence is not a determinant of polysome association. An undermethylated cap 4 structure was present on substrate and mRNA spliced leaders in nt 20-29 mutated exons; nt 20-29 mRNA was not present in polysomes. A differential pattern of cap 4 methylation was seen between the nt 10-19 substrate spliced leader RNA and the nt 10-19 spliced leaders found in the poly(A)+ population of RNA; the nt 10-19 mRNA was not seen in polysomes. Undermethylated spliced leaders did not associate efficiently with polysomes, suggesting a requirement for the cap 4 and/or primary sequence of the spliced leader in translation. This is the first report demonstrating that the spliced leader contains critical structural or sequence determinants for association with polysomes and, hence, translation.
Insights
In kinetoplastids, the spliced leader sequence is crucial for mRNA translation. Mutations affecting cap 4 methylation prevent polysome association, highlighting its role in protein synthesis.
Area of Science:
- Molecular Biology
- Genetics
- Parasitology
Background:
- Kinetoplastid mRNAs possess a conserved 39-nucleotide spliced leader (SL) at their 5'-terminus.
- This SL is added via trans-splicing and is modified with a cap 4 structure, essential for mRNA function.
- Previous studies indicated SL nucleotides 10-39 were not essential for SL RNA transcription or trans-splicing.
Purpose of the Study:
- To investigate the role of specific spliced leader sequences and their methylation patterns in mRNA translation.
- To determine if the spliced leader sequence influences polysome association, a key step in protein synthesis.
Main Methods:
- Mutagenesis of the spliced leader sequence in Leishmania tarentolae.
- Assaying polysome association of mutated mRNAs using ultracentrifugation.
- Analyzing the methylation status of the cap 4 structure on spliced leader RNAs and mRNAs.
Main Results:
- Mutations in the SL nucleotides 20-29 and 10-19 resulted in undermethylated cap 4 structures and prevented polysome association.
- The SL nucleotides 28-39 region, despite conserved primary sequence, did not affect polysome association.
- Undermethylated SLs showed inefficient polysome binding, suggesting a requirement for cap 4 methylation or specific SL sequences.
Conclusions:
- The spliced leader sequence, particularly its cap 4 methylation, is critical for efficient mRNA translation in kinetoplastids.
- This study identifies the spliced leader as a key determinant for polysome association and protein synthesis initiation.