Single nucleotide resolution of promoter activity and protein binding for the Leishmania tarentolae spliced leader RNA gene

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Abstract

In Kinetoplastid protozoa, trans-splicing is a central step in the maturation of nuclear mRNAs. In Leishmania, a common 39 nt spliced-leader (SL) is transferred via trans-splicing from the precursor 96 nt SL RNA to the 5′ terminus of all known protein-encoding RNAs. In this study, promoter elements of the L. tarentolae SL RNA gene have been identified with respect to transcriptional activity and putative transcription factor binding. We have mapped the essential regions in the SL RNA gene promoter at single nucleotide resolution using both in vivo transcription and in vitro protein/DNA binding approaches. Two regions located upstream of the SL RNA gene were identified: a GN3CCC element at −39 to −33 and a GACN5G element at −66 to −58 were essential for SL RNA gene transcription in stably transfected cells. Consistent with other known bipartite promoter elements, the spacing between the GN3CCC and GACN5G elements was found to be critical for proper promoter function and correct transcription start point selection, as was the distance between the two elements and the wild-type transcription start point. The GACN5G element interacts specifically and in a double-stranded form with a protein(s) in Leishmania nuclear extracts. The degree of this protein–DNA interaction in vitro correlated with SL RNA gene transcription efficiency in vivo, consistent with a role of the protein as a transcription factor. The core nucleotides GACN5G fit the consensus PSE promoter structure of pol II-transcribed snRNA genes in metazoa.

Introduction

Protozoa of the order Kinetoplastida, Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., cause afflictions such as African sleeping sickness, Chagas disease and leishmaniasis [1] and have complex developmental life cycles in their mammalian and insect hosts. It is likely that regulated gene expression determines the levels of stage-specific proteins. Although most of this regulation appears to be post-transcriptional, one aspect of gene expression, transcription initiation, has been the subject of considerable research in trypanosomes, but is still poorly understood [2]. Very few promoters for protein-encoding genes have been identified in trypanosomes. Their location has been obscured due to the occurrence of polycistronic transcription and trans-splicing of pre-mRNAs 3, 4. The promoters for variant surface glycoprotein (VSG) and procyclin genes of T. brucei are the best studied for protein-encoding genes in this class of organisms (reviewed in [4]). These promoters, however, direct transcription by an RNA polymerase I (pol I)-like polymerase [5], versus the typical pol II transcription of housekeeping genes [6]. The best characterized pol II promoter for protein-encoding genes in trypanosomes has been located by transient and stable transfection in the 0.7 kb region upstream of the hsp70 gene 3 of T. brucei [7].

The spliced-leader (SL), or mini-exon, RNA gene is transcribed from a pol II promoter in T. brucei 5, 8 and Leishmania tarentolae [9], although this characterization is controversial due to additional evidence from T. brucei 10, 11 suggesting that pol III is involved. The possibility that the SL RNA gene, like the vertebrate U6 RNA gene, is transcribed by a polymerase using sequence elements that share characteristics of both pol II and pol III promoters has also been suggested [12]. Further study of the promoter architecture and transcription factor involved in the SL RNA gene transcription is necessary to determine whether conventional rules of gene transcription apply in the trypanosomes.

The SL RNA is involved in trans-splicing, an RNA processing pathway shared by members of the Kinetoplastida [13]. This bimolecular reaction involves the ligation of a spliced-leader to pre-mRNA via a 2-step phosphodiester exchange reaction and generates a capped 5′ terminus on mature mRNA [14]. The 39 nt SL possesses a ‘cap 4’ structure at its 5′ terminus and is transcribed independently from the protein-coding genes as a discrete molecule, the SL RNA 15, 16, 17.

Like the metazoan U6 RNA genes, transcription of the Kinetoplastid SL RNA gene is dependent on upstream sequences [4]. In transfection assays, positions −70 to −30 relative to the transcription start point (TSP) of the L. amazonensis gene [18], positions −81 to −72 of the T. brucei gene [12], and positions −53 to −40 of the T. cruzi gene [19] are essential for episomal SL RNA gene transcription. Mutation of positions −61 to −55 and −41 to −31 reduce transcription of the T. brucei SL RNA gene both in vivo and in vitro [12]. The Leptomonas seymouri gene is thought to possess three elements, −70 to −51, −40 to −31, and −10 to −1, that are essential for SL RNA synthesis in vivo [20]. Protein binding is observed for the T. cruzi element [19] and for two elements in L. seymouri 20, 21. In previous studies, SL RNA gene promoter elements were defined by block mutagenesis. As a consequence, the sizes of essential sequences for SL RNA gene transcription range from 16 bp in T. cruzi [19] to 40 bp in L. amazonensis [18]. Promoter elements are located only upstream of the SL RNA gene 12, 20, 22.

Previously, we demonstrated in L. tarentolae that a 10 bp block conserved among a spectrum of human pathogenic Leishmania species at positions −67 to −58 [23] is essential for SL RNA synthesis [9]. Herein, using a saturating mutagenesis approach, we show that the L. tarentolae SL RNA gene promoter consists of two elements, and define each element at the resolution of single nucleotides. The element spanning position −67 to −58 bears an essential core sequence of GACN5G. Core nucleotides in the second element from −40 to −31 are GN3CCC. A DNA-binding activity present in L. tarentolae nuclear extracts is specific for the −67 to −58 region. We find a correlation between the degree of protein binding in vitro and efficiency of SL RNA synthesis in vivo, indicating that each nucleotide necessary for protein binding is necessary for promoter activity; nucleotides that can be mutated without affecting protein binding are not required for promoter activity.

Section snippets

Site-directed mutagenesis

The linker scan mutations were constructed from positions −80 to −31 using the Sculptor Mutagenesis kit (Amersham) on an M13 template. Complimentary oligonucleotides were used to introduce the 10 bp block mutations (5′-CCTCGAGGAA-3′) that contain an XhoI site [9]. These XhoI sites were used subsequently in constructing spacing mutants (see Section 2.2). Site-directed mutagenesis of the −40/−31 region was performed in M13 by the dut/ung method as described previously [24]. Plaques were picked

A second element is essential for SL RNA gene expression in L. tarentolae

A highly conserved sequence block in the region −67 to −58 relative to the TSP [23] of the L. tarentolae SL RNA gene [29] was identified previously as a promoter element [9]. In other Trypanosomatids, the upstream region −80 to −30 has been reported to contain either one 18, 19 or two 12, 20 discrete promoter elements. To test whether a second upstream element is present in L. tarentolae, we examined a linker scan mutation series from −80 to +19 relative to the SL RNA gene TSP.

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Discussion

We have performed a detailed functional analysis of the L. tarentolae SL RNA gene promoter and identified eight core nucleotides. Two essential elements, GN3CCC located at −39 to −33 and GACN5G located at −66 to −58, were found to be required for efficient SL RNA gene transcription in vivo. Using the gel shift assay, a protein component in L. tarentolae nuclear extract was shown to specifically recognize the double-stranded GACN5G element. Each point mutation in the GACN5G element that greatly

Acknowledgements

We thank Steve Beverley for the pX plasmid, and the laboratories of Larry Feldman, Patricia Johnson, and Olaf Schneewind. We also thank Dan Ray for critical comments. This work was supported by NIH grant AI34536. N.R.S. and R.M.S. were supported by an NIH training grant in Microbial Pathogenesis 2-T32-AI-07323.

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      More recent studies have explored the implications of these findings and demonstrated that formation of the cap early in transcription is mediated by recruitment of the capping machinery to the phosphorylated carboxyl-terminal domain of the largest subunit of RNA polymerase II (15-18). At present the RNA polymerase transcribing the SL RNA gene has not been identified unambiguously, although there is a strong suggestion that it might be RNA polymerase II (19, 20). It is conceivable that, similar to other systems, there is a specific interaction between the capping machinery and the SL RNA transcription apparatus, either through the RNA polymerase large subunit or another component of the transcriptional machinery.

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    Present address: Department of Genetics, Washington University, St. Louis, MO 63110.

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