Two functionally dependent acetylcholine subunits are encoded in a single Caenorhabditis elegans operon

Abstract

The deg-3 gene from the nematode Caenorhabditis elegans encodes an alpha subunit of a nicotinic acetylcholine receptor that was first identified by a dominant allele, u662, which produced neuronal degeneration. Because deg-3 cDNAs contain the SL2 trans-spliced leader, we suggested that deg-3 was transcribed as part of a C. elegans operon. Here we show that des-2, a gene in which mutations suppress deg-3(u662), is the upstream gene in that operon. The des-2 gene also encodes an alpha subunit of a nicotinic acetylcholine receptor. As expected for genes whose mRNAs are formed from a single transcript, both genes have similar expression patterns. This coexpression is functionally important because (i) des-2 is needed for the deg-3(u662) degenerations in vivo; (ii) an acetylcholine-gated channel is formed in Xenopus oocytes when both subunits are expressed but not when either is expressed alone; and (iii) channel activity, albeit apparently altered from that of the wild-type channel, results from the expression of a u662-type mutant subunit but, again, only when the wild-type DES-2 subunit is present. Thus, the operon structure appears to regulate the coordinate expression of two channel subunits.

Figure 1

The des-2 deg-3 operon. (A) Genomic organization. Arrows mark the site of spliced leader addition. Although the longer des-2 cDNA lacks a spliced leader, it starts at the same place as SL1-spliced variants that were identified from a random primed cDNA library. deg-3 is trans-spliced to the SL2 leader (7) (B) Alignment of DES-2 and DEG-3. The positions of the des-2 mutations (all of which result in premature termination) are indicated by asterisks. Codon changes are: hm5 (TGG to TGA), hm6 (TGG to TAG), and u695 (CAG to TAG). Thin underlining indicates the neurotransmitter gated-channel signature (24) and thick underlining indicates the putative transmembrane domains (see ref. 7). The two adjacent cysteines indicative of nAChR α subunits (25) are at amino acids 220 and 221 in DES-2.

Figure 2

Expression of a des-2 lacZ fusion. This pattern is similar to that for deg-3 expression (7). Identified cells include the M1 head muscles (the staining in front of the IL2 neurons; these cells were identified by their structure in a des-2 gfp expressing strain), IL2 neurons (these cells disappear in unc-86 animals), FLP neurons (these cells disappear in mec-3 animals), PVD neurons (identified by the pattern of their processes using a des-2 gfp fusion and because they disappear in mec-3 and unc-86 animals), and PVC neurons (these cells disappear in deg-1 animals).

Figure 3

ACh response of the normal and mutant DES-2/DEG-3 channel. Representative current responses were elicited by 1 mM ACh in voltage-clamped oocytes during the time indicated by the solid bar.