Kagoshima H et al. (1998) Worm Breeder's Gazette "The (only?) C.elegans runt gene"

Burglin TR, Kagoshima H

[Worm Breeder's Gazette, 1998]

The Runt gene family is a new family of heteromeric DNA binding transcription factors. Recent work has revealed that members of this family play  important roles in embryonic development of sea urchin, segmentation of Drosophila, and hematopoiesis and osteoblast differntiation in mouse and human. The heteromeric complex consists of two subunits, a DNA binding subunit aand its partner subunit b. The a subunit binds directly to a specific DNA sequence (PuCCPuCAG [Pu=A/G]) while theb subunit associates with ato form a heterodimer that has a greater affinity for DNA. The asubunit shares a region of high homology, termed the Runt domain (Fig. 1), with the products of the Drosophila segmentation gene runt and the human acute myeloid leukemia-related gene AML1. A series of biochemical analyses revealed that the Runt domain is sufficient for both functions of the asubunit: DNA binding and heterodimerization with the bsubunit. In mammals, there are 3 types of asubunits, aA,aB and aC. Targeted disruption of aA in mouse results in a complete lack of ossification in their skeletal systems. The human homolog of aB, AML1, is known as the most frequent target of chromosomal translocations associated with leukemia, and targeted mice lacking AML1 showed no fetal liver hematopoiesis. aB has thus proved to be essential for hematopoiesis of all lineages. We found a C. elegans homolog of runt by searching in the C. elegans databases, which we propose to call rnt-1. We made a promoter-gfp-lacZ reporter construct to examine the temporal and spatial expression pattern.  We observed gfp expression in the hypodermis, the seam and probably their precursor cells starting from the 2-fold stage embryo to adult. This result implied that rnt-1 might have quite different functions in different organisms, which contrasts with the high sequence conservation. Moreover, so far  no bsubunit gene has been found in C. elegans; this subunit has important regulatory functions for the Runt domain. To elucidate the function of rnt-1 in worms, we are going to do RNAi experiments and screen knock-out mutant libraries. This should give us new insights into the function of this gene family.  

Hope IA (1993) Worm Breeder's Gazette "Gene with Early, Cell Lineage Specific Expression is Homologous to Fork Head"

Hope IA

[Worm Breeder's Gazette, 1993]

The plasmid pUL#24 C7was identified in my initial promoter trap screen (Hope, Development, 113, 399) and consists of a 6.5kb C.elegans genomic DNA fragment cloned upstream of lacZ in Andy Fire's vector, pPD22 .11.C.elegans transformants containing this DNA express -galactosidase during early embryogenesis. -galactosidase was detected in: i. A subset of the AB ll lineage, descendants of ABalpa, ABalpp, ABaIaa, ABplaa, ABplpa, ABpraa and ABpIpa, from the 5th cell division in the AB cell lineage, for approximately 3 cell generations. ii. All of the D cell lineage, from the 2nd cell division in the D cell lineage until shortly after the 4th cell division. iii. Z1 and Z4 from midembryogenesis until shortly after hatching. The gene with which lacZ has been fused in pUL#24 C7has been called pes-1 (pattern expression site). Genomic DNA sequencing has revealed that the lacZ gene has been fused to DNA encoding part of a protein domain homologous to the DNA binding domain of the fork head family of transcription factors. Sequence 5' from the point of fusion with lacZ was determined from pUL#24 C7.Sequence 3' from the point of fusion was obtained by subcloning from the cosmid T28H11 (thanks Alan and Ratna) which contains genomic DNA flanking the insert of pU#24C7. The predicted PES1 protein shows 44% amino acid identity to fork head over a 95 residue region. Two other C.elegans genes have been described which have homology to the fork head family (WBG12.3, 37 and 12.4, 32). [See Figure 1] PCR, using a primer for the trans-spliced leader SL1 ,was used to examine transcripts emanating from pes-1 .There appear to be 3 sites at which transcription can initiate. Two of the transcripts would encode different proteins although both would contain the predicted DNA binding domain. The third transcript does not appear to contain a codon which would initiate translation through the fork head homologous domain, but this needs further study. It is intriguing that there are 3 components to the -galactosidase expression pattem and 3 different transcripts have been detected. The possibility that a different transcript is produced in each founder cell lineage is being examined. pUL#24 C7was originally selected because the -galactosidase expression pattem suggested the gene tagged with lacZ was under the control of a regulatory system important in early embryogenesis. The possibility that pes-1 encodes a specific DNA binding protein means there is interest not only in how pes-1 is regulated but also in what pes-1 is regulating.

Conrad RC et al. (1992) Worm Breeder's Gazette "A Synthetic Sequence Can Replace the rol-6 Outron to Allow SL1-Specific Trans-Splicing"

Blumenthal TE, Conrad RC

[Worm Breeder's Gazette, 1992]

We have been working on the problem of what signals an initial transcript to be trans-spliced. Our previous results (Conrad et al. 1991,Mol. Cell. Biol. 11:1921) indicated that a transcript that begins with an intron, rather than the first exon, is trans-spliced to SL1 ,and that no sequence-specific information is required for SL1 splicing. We named this 5' intron an "outron". The requirement for the splice-acceptor site at the outron/exon border is a close match to the consensus: UUUCAG/Pu. Our interest now is in what is required upstream of the splice site. Our initial hypothesis is that both AU-richness and a minimum length are the primary, and perhaps sole, requirements. This expectation arises from several observations: 1) our previous results showed that an intron contains all the information necessary to signal trans-splicing, 2) no consensus sequences can be discerned from gazing at the many known outron sequences, and 3) there is a perfect splice-acceptor site in the 5' UTR of act-4 which is not trans-spliced, and its 5' UTR is only 33 nt and ~50% AU (functional outrons and introns are around 70% AU). To test the hypothesis we are creating a set of synthetic outrons to replace the rol-6 outron. These constructs can then be used to transform worms, and the resultant RNAs analyzed to determine if the particular potential outron is functional for trans-splicing. We deleted all but the six nucleotides at the 5' end and the six at the 3' end of the (normally 173 nt) rol-6 outron. This preserves (we hope) the transcriptional start site and splice acceptor site. In place of the 161 deleted bases we inserted a 15 nt sequence containing a unique XhoI site. We called this construct OM (for minimal outron). On assaying this construct using RNA PCR, we found that its RNA product was not trans-spliced. Further constructs were created using an oligonucleotide duplex as an insert into the XhoI site. The insert was 25 nt long, with the repeated sequence A2U4 in one orientation ("[U]") and U2A4 in the other ("[A]"). A triple-insert, S'[U][U][A]3', gave strong trans-splicing, with little unspliced product accumulating. Single inserts (both [A] and [U]) also were trans-spliced, but unspliced product also accumulated,so we presume the trans-splicing was less efficient with these shorter outrons. A 5'[U][U]3' double-insert gave an intermediate trans-spliced-to-unspliced ratio. Almost all trans-splicing was to SL1 ,which provides the strongest support yet that SL1 splicing is the default mode. We conclude that SL1 trans-splicing can be signalled simply by the presence at the 5' end of a pre-mRNA of an AU-rich sequence followed by a splice-acceptor site. A final point is that all of these constructs had to be co-injected with a wild-type rol-6 construct (pRF5 ).No stable roller strains were obtained with any of the OM-derived constructs, although roller F1 swere. (We can distinguish the gene products of pRF5 and the OM constructs because the latter were marked by a set of codon-conserving nucleotide changes, a 5-base change in a 7-base stretch). This raises some interesting questions about a possible role for the portion of rol-6 that contains the outron sequence in promoter function or in stabilization of the pre-mRNA.