Elsevier

Gene

Volume 311, 5 June 2003, Pages 59-69
Gene

Drosophila lola encodes a family of BTB-transcription regulators with highly variable C-terminal domains containing zinc finger motifs

https://doi.org/10.1016/S0378-1119(03)00554-7Get rights and content

Abstract

Alternative splicing is an important mechanism contributing to the increased proteome diversity in higher eukaryotes. We have explored the alternative splicing events in the Drosophila longitudinals lacking (lola) gene by means of 5′ RACE, 3′ RACE, genome sequence searches, and EST sequencing. We demonstrated that the lola locus is comprised of 32 exons spanning over 60 kb, and encodes a total of 80 alternatively spliced variants consisting of 5′ and 3′ variable sequences and constitutive common exons. All the variants shared a common sequence (exons 5–8) encoding the N-terminal region containing the BTB domain, but both the 5′ and 3′ ends were variable. There were four promoters responsible for the variation in the 5′ end (exons 1–4). Alternative splicing was involved in the variation in the 3′ end corresponding to the C-terminal variable region, which was encoded by one or two exons that were selected from 20 groups of exons in a mutually exclusive manner (exons 9–32). Seventeen of the 20 isoforms contained C2H2-like zinc finger motifs in the C-terminal variable region. Analyses of the 3′ variant-specific cDNA pools revealed that all combinations of 5′ and 3′ variable sequences were expressed in both the embryonic and third instar larval stages. Since the BTB domain mediates dimerization, lola encodes a family of transcription regulators with a large variety of DNA- or protein-binding specificities, and could be involved in various developmental processes, including the embryonic neural pathfindings. We also showed that the structures of Lola isoforms were highly conserved in Drosophila pseudoobscura.

Introduction

The human genome sequence contains approximately 32,000 genes, only twice as many as that of Caenorhabditis elegans or Drosophila melanogaster (International Human Genome Sequencing Consortium, 2001). Therefore, the complexity of organisms is not simply achieved by an increase in the number of genes. They seem to utilize a single gene in a variety of ways, through co- or post-transcriptional mechanisms, which include multiple transcription start or termination sites, alternative splicing and RNA editing. Among them, alternative splicing is rather common in higher eukaryotes (Graveley, 2001). It has been estimated that 40–60% of human genes have at least one alternatively spliced form (Modrek and Lee, 2002). However, the mechanisms of alternative splicing have only been elucidated for a limited number of genes (López, 1998; Smith and Valcárcel, 2000).

A family of transcription factors containing the BTB domain and zinc finger motif are involved in a wide variety of developmental pathways and often produce multiple isoforms via alternative splicing. In Drosophila, the tramtrack (ttk) gene is required for development of the nervous system (Guo et al., 1995). Two alternatively spliced isoforms that share a common N-terminal region, including the BTB domain, have distinct C-terminal regions, each with a unique pair of ‘classical’ TFIIA-type C2H2 zinc fingers that recognize different DNA sequences (Read and Manley, 1992). The Broad Complex (BR-C) gene belonging to the same family is one of the few genes directly induced by the ecdysteroid hormone, and regulates the expression of subordinate genes involved in the metamorphosis. There are four isoform-specific exons encoding pairs of classical C2H2 zinc finger motifs (DiBello et al., 1991). The modifier of mdg4 (mod(mdg4)) gene, also known as E(var)3-93D, is involved in a variety of processes, such as silencing in position effect variegation, control of the gypsy insulator sequence, regulation of homeotic gene expression, and programmed cell death (Dorn et al., 1993; Gerasimova and Corces, 1998; Harvey et al., 1997). The mod(mdg4) gene produces 26 different isoforms generated by alternative splicing (Büchner et al., 2000; Dorn et al., 2001). The common exons encoding the BTB domain are alternatively spliced to one of the 3′-variable exons encoding a modified version of C2H2 that potentially forms a finger structure (Büchner et al., 2000; Dorn et al., 2001).

The longitudinals lacking (lola) gene is another member of the BTB-zinc finger protein genes and has been shown to be required for proper pathfinding and target recognition of the SNb motor axons in the Drosophila embryo (Crowner et al., 2002; Giniger et al., 1994; Madden et al., 1999). Giniger et al. (1994) described two transcripts, lola 3.8 and 4.7, which were generated from the lola locus by the use of two promoters and alternative splicing. Two transcripts encode the isoforms sharing a large common N-terminal region, including the BTB domain, and a C-terminal region unique to each isoform. The C-terminal region of the long isoform encoded by lola 4.7 contains a pair of classical C2H2 zinc finger motifs, with a minor modification: the second histidine in the first finger is replaced by cysteine (hereafter, referred as C2HC–C2H2 motif). The shorter isoform lola 3.8 does not encode zinc finger motifs or any other known motifs.

Here, we show that lola is a large and complex locus, extending over 60 kb, and encodes a total of 80 variants through multiple promoter activity and alternative splicing. Lola isoforms consist of a BTB-containing N-terminal region and a C-terminal variable region encoded by alternatively spliced exons among 20 groups, 17 of which have a unique zinc finger motif. Since the BTB domain mediates dimerization, lola could theoretically encode a family of transcription regulators with a large variety of DNA- or protein-binding specificities.

Section snippets

EST clones

The BDGP EST database (http://www.fruitfly.org/) was searched for cDNA clones that matched lola cDNA (Giniger et al., 1994; Toba et al., 1999) as queries. Five clones, LD03274, LD11722, LD13249, LD15869 and GM01463 were obtained from the BDGP. The cDNA inserts were sequenced using a BigDye terminator cycle sequencing kit (Perkin–Elmer) and a Perkin–Elmer ABI PRISM genetic analyzer 310.

RACE

The 5′ ends of the lola transcripts were determined using the 5′ rapid amplification of cDNA ends (RACE)

cDNAs of lola

The gene misexpression system involving modified P-elements is widely used in Drosophila as a tool for identifying new genes. We identified lola as a gene whose misexpression could induce homeotic transformation of aristae to tarsi (Toba et al., 1999). Molecular analysis of forcibly expressed transcripts suggested that lola produced multiple forms of transcripts that appeared to be generated through alternative splicing. To reveal the variability of the lola transcripts, we sequenced five EST

lola encodes a large family of alternatively spliced transcripts

We have explored the variability of the Drosophila lola cDNA structure by means of 5′ RACE, 3′ RACE, genome sequence searches, and EST sequencing, and demonstrated that the lola locus is comprised of 32 exons spanning over 60 kb, and encodes a total of 80 alternatively spliced variants consisting of 5′ and 3′ variable sequences and constitutive common exons. The common exons encode the N-terminal region containing the BTB domain, and the variable exons encode the C-terminal variable domain,

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (C) ‘Genome Science’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan (#12202002).

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    Present address: Takashi Ohsako, Drosophila Genetic Resource Center, Kyoto Institute of Technology, Kyoto 616-8354, Japan.

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