Functional analysis of putative operons in Brugia malayi

Abstract

Operons are a common mode of gene organization in Caenorhabditis elegans. Similar gene arrangements suggest that functional operons may exist in Brugia malayi. To definitively test this hypothesis, a bicistronic reporter vector consisting of an upstream firefly luciferase gene and a downstream renilla luciferase gene was constructed. The genome was then surveyed to identify 15 gene pairs that were likely to represent operons. Two of four domains upstream of the 5′ gene from these clusters exhibited promoter activity. When constructs replicating the promoter and intergenic arrangement found in the native putative operon were transfected into embryos, both firefly and renilla activities were detected, while constructs with the promoter alone or intergenic region alone produced no activity from the downstream reporter. These data confirm that functional operons exist in B. malayi. Mutation of three U-rich element homologues present in one of the operons resulted in a decrease in downstream renilla reporter activity, suggesting that these were important in mRNA maturation. Hemi-nested reverse transcriptase-PCR assays demonstrated that while the mRNA encoding the native downstream open reading frame of one operon contained an SL1 spliced leader at its 5′ end, the renilla gene mRNA produced from the corresponding transgenic construct did not.

Introduction

Filarial parasites represent a significant public health problem throughout much of the developing world (Michael et al., 1996). There is currently a lack of effective drugs to treat these infections, making studies to identify new chemotherapeutic candidates a priority. As a result, studies of the basic biology of these organisms, and in particular studies of biological processes that do not appear to be shared between the parasite and its human host, are of particular importance.

During the past decade, it has become clear that many of the genes of the free-living nematode Caenorhabditis elegans are organized into operons (Zorio et al., 1994, Blumenthal et al., 2002). This type of gene arrangement, while common among prokaryotes, appears to be confined to certain groups of eukaryotes. Most operons in C. elegans contain a small number of genes (mean 2.6) (Blumenthal et al., 2002), although the maximum number of genes found to be arranged in an operon is eight (Blumenthal et al., 2002). In C. elegans operons, transcription proceeds from a single promoter which is located upstream of the first gene in the operon (Spieth et al., 1993). Transcription proceeds through the upstream gene and then through a short intergenic spacer domain into the downstream genes, resulting in a polycistronic transcript. The resulting polycistronic pre-mRNA is then resolved into mono-cistronic mature mRNAs. This occurs through the process of trans-splicing. In C. elegans operons, the mature mRNA of the gene located at the 5′ end of these clusters receives a spliced leader (SL) known as SL1, while the genes located downstream in the polycistronic pre-RNA are usually matured by the addition of SL2 sequences (Spieth et al., 1993). However, the SL1 can substitute for the SL2 in the maturation of some downstream mRNAs, particularly in operons where the intergenic region is large (Graber et al., 2007). Thus, identification of bona fide operons in C. elegans is facilitated by experiments that look for the presence of SL2 sequences at the 5′ end of the mature mRNAs of genes whose function and genomic arrangement suggest that they might be part of an operon (Blumenthal et al., 2002).

The maturation of the polycistronic mRNAs produced from operons is somewhat complicated by the fact that this process would be expected to result in the production of an uncapped 5′ end on the mRNA intermediate containing the downstream gene, something that normally would mark it for rapid degradation in the cell. This difficulty is overcome by the interaction of a special small nuclear ribonucleoprotein (SnRNP) containing the SL2 RNA with sequences encoded in the intergenic region, resulting in the addition of an SL2 to the downstream intermediate and the production of a mature SL2 containing mRNA (MacMorris et al., 2007). This process is mediated by a U-rich (Ur) domain encoded in the intergenic region (Huang et al., 2001). The Ur domain consists of a motif (with a consensus sequence of either UAUUUU or UUUUAU), that is generally located approximately 27 nucleotides (nt) downstream from the poly A addition signal of the upstream gene in the operon (Graber et al., 2007).

Analysis of the genomic sequence of the human nematode filarial parasite Brugia malayi has suggested that, like C. elegans, many of the genes in this parasite might also be arranged into operons (Guiliano and Blaxter, 2006, Ghedin et al., 2007). However, the presence of functional operons in B. malayi has been difficult to confirm. This is because in these parasites only the SL1 sequence is found on all mRNAs. Thus, any downstream genes in a putative operon would probably also receive an SL1 sequence at their 5′ ends, making them experimentally indistinguishable from genes which are transcribed from their own promoter. One study has reported detecting an mRNA species spanning the two open reading frames (ORFs) of genes in a putative operon of B. malayi using reverse transcriptase-PCR (RT-PCR) (Guiliano and Blaxter, 2006). However, as pointed out by Blumenthal (2004) such RNAs encompassing two adjacent genes might represent dead-end products and not physiologically relevant pre-mRNAs. Thus, proving that putative operon structures represent bona fide operons requires that one demonstrate the intergenic region does not contain a promoter, and that all transcription originates from the region upstream of the 5′-most gene of the cluster.

A transient transfection system based upon biolistic bombardment of isolated B. malayi embryos has been used to study promoter structure and trans-splicing in this human filarial parasite (Higazi et al., 2002, Higazi et al., 2005, Shu et al., 2003, Higazi and Unnasch, 2004, Liu et al., 2007, Liu et al., 2009, Oliveira et al., 2008). We hypothesized that this method might also be used to test the hypothesis that operons exist in B. malayi. To accomplish this, a bicistronic reporter vector was constructed that included two reporter genes. This vector could then be used to prepare constructs that recapitulated the structure of a putative operon, but in which the upstream and downstream native ORFs were replaced by the separately assayable reporters. Here we report the results obtained in using this reporter vector to test the hypothesis that operons exist in B. malayi, and to begin to examine the mechanisms involved in processing of nascent mRNAs generated from one such synthetic operon construct.