The promoter and transcribed regions of the Leishmania tarentolae spliced leader RNA gene array are devoid of nucleosomes.
BACKGROUND
The spliced leader (SL) RNA provides the 5' m7G cap and first 39 nt for all nuclear mRNAs in kinetoplastids. This small nuclear RNA is transcribed by RNA polymerase II from individual promoters. In Leishmania tarentolae the SL RNA genes reside in two multi-copy tandem arrays designated MINA and MINB. The transcript accumulation from the SL promoter on the drug-selected, episomal SL RNA gene cassette pX-tSL is ~10% that of the genomic array in uncloned L. tarentolae transfectants. This disparity is neither sequence- nor copy-number related, and thus may be due to interference of SL promoter function by epigenetic factors. To explore these possibilities we examined the nucleoplasmic localization of the SL RNA genes as well as their nucleosomal architecture.


RESULTS
The genomic SL RNA genes and the episome did not co-localize within the nucleus. Each genomic repeat contains one nucleosome regularly positioned within the non-transcribed intergenic region. The 363-bp MINA array was resistant to micrococcal nuclease digestion between the -258 and -72 positions relative to the transcription start point due to nucleosome association, leaving the promoter elements and the entire transcribed region exposed for protein interactions. A pattern of ~164-bp protected segments was observed, corresponding to the amount of DNA typically bound by a nucleosome. By contrast, nucleosomes on the pX-tSL episome were randomly distributed over the episomal SL cassette, reducing transcription factor access to the episomal promoter by approximately 74%. Cloning of the episome transfectants revealed a range of transcriptional activities, implicating a mechanism of epigenetic heredity.


CONCLUSIONS
The disorganized nucleosomes on the pX episome are in a permissive conformation for transcription of the SL RNA cassette approximately 25% of the time within a given parasite. Nucleosome interference is likely the major factor in the apparent transcriptional repression of the SL RNA gene cassette. Coupled with the requirement for run-around transcription that drives expression of the selectable drug marker, transcription of the episomal SL may be reduced even further due to sub-optimal nucleoplasmic localization and initiation complex disruption.
Hitchcock
Robert A
RA
Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095-1489, USA. mrbob@ucla.edu <mrbob@ucla.edu>
Thomas
Sean
S
Campbell
David A
DA
Sturm
Nancy R
NR
D000818:Animals; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence; D007891:Leishmania / Q000235:genetics* / Q000378:metabolism; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020038:RNA, Spliced Leader / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D009707:Nucleosomes; D020038:RNA, Spliced Leader