Neuron-type specific regulation of a 3'UTR through redundant and combinatorially acting cis-regulatory elements

Abstract

3' Untranslated region (UTR)-dependent post-transcriptional regulation has emerged as a critical mechanism of controlling gene expression in various physiological contexts, including cellular differentiation events. Here, we examine the regulation of the 3'UTR of the die-1 transcription factor in a single neuron of the nematode C. elegans. This 3'UTR shows the intriguing feature of being differentially regulated across the animal's left/right axis. In the left gustatory neuron, ASEL, in which DIE-1 protein is normally expressed in adult animals, the 3'UTR confers no regulatory information, while in the right gustatory neuron, ASER, where DIE-1 is normally not expressed, this 3'UTR confers negative regulatory information. Here, we systematically analyze the cis-regulatory architecture of the die-1 3'UTR using a transgenic, in vivo assay system. Through extensive mutagenesis and sequence insertions into heterologous 3'UTR contexts, we describe three 25-base-pair (bp) sequence elements that are both required and sufficient to mediate the ASER-specific down-regulation of the die-1 3'UTR. These three 25-bp sequence elements operate in both a redundant and combinatorial manner. Moreover, there are not only redundant elements within the die-1 3'UTR regulating its left/right asymmetric activity but asymmetric 3'UTR regulation is itself redundant with other regulatory mechanisms to achieve asymmetric DIE-1 protein expression and function in ASEL versus ASER. The features of 3'UTR regulation we describe here may apply to some of the vast number of genes in animal genomes whose expression is predicted to be regulated through their 3'UTR.

FIGURE 1.

Laterality of the ASE neurons. (A) Model of gene regulatory network responsible for the generation of ASE asymmetry (Hobert 2006). (B) mir-273 locus and deletion allele tested in this analysis. The deletion allele is from Miska et al. (2007). (C) mir-273 null mutant animals do not have an ASE asymmetry phenotype. lsy-6 loss-of-function and control data taken from Johnston and Hobert (2003). (D) Alignment of the C. elegans die-1 3′UTR with three other related nematode species. Multiple sequence alignments were generated with T-coffee (Notredame et al. 2000). Only the first 400 bp of the die-1 3′UTR is shown because it contains all regulatory information (see Fig. 3). Gray boxes indicate the most conserved, extended sequenced patches, both of which show matches to the mir-273 family of miRNAs (for miRNA/target pairing, see Supplemental Fig. 1). Sequence elements identified by mutational analysis motifs to be involved in 3′UTR regulation are colored. Red lines indicate the extent of the 25-bp scanning mutagenesis windows (Supplemental Figs. 2, 3).

FIGURE 2.

Features of the die-1 3′UTR sensor system. (A) Schematic of the die-1 3′UTR sensor and gfp images that show expression of the sensor in transgenic adult worms (all scoring throughout this article is done in young adults). ASEL and ASER are labeled by red circles. AWC neurons that serve as internal control are out of the plane of view. The data on unc-54 and cog-1 have been described before and are taken from Didiano and Hobert (2006). (B) Quantification of 3′UTR sensor expression. Each set of three bars represents an independent transgenic line. Individual animals are scored and binned into one of three categories: gfp expression in ASEL > ASER (left black bar), ASEL = ASER (middle gray bar) or ASER > ASEL (right black bar). Multiple transgenic lines of the same construct are shown for unc-54 and die-1 3′UTRs. Constructs were injected at 5 ng/μL. Injection of the wild-type die-1 3′UTR sensor construct at high concentrations (100 ng/μL) results in a loss of the observed regulation. The unc-54 data were freshly rescored. (C) die-1 3′UTR regulation is dependent upon ASE asymmetry. An integrated die-1 3′UTR sensor construct (otIs260) displays slightly higher levels of regulation than the extrachromosomal lines shown in panel B. Regulation of the integrated die-1 3′UTR sensor construct is lost in 2-ASER (lsy-6) or 2-ASEL (ceh-36∷lsy-6) mutants. The lsy-6 allele used (ot71) has a 100% 2-ASER phenotype (Johnston and Hobert 2003). The ceh-36∷lsy-6 strain otIs204 expresses lsy-6 bilaterally in ASEL and ASER, causing a near 100% 2-ASEL phenotype, as previously described (Ortiz et al. 2009).

FIGURE 3.

Mutational analysis of the die-1 3′UTR. Primary scoring data for multiple transgenic lines is shown, which is translated into the symbols ++, +, +/−, and −, which are used to indicate the levels of differential regulation of gfp expression in ASEL versus ASER (“asymmetric regulation”). These categories are based on asymmetry indices (A.I.s) calculated for each transgenic line, as described in the Materials and Methods.

FIGURE 4.

Identification of three cis-regulatory elements required for asymmetric regulation of the die-1 3′UTR in ASEL versus ASER. For explanation of the symbols, see the Figure 3 legend. Gray boxes are the conserved mir-273 sites shown in Figure 1C.

FIGURE 5.

die-1 3′UTR sequence motif replacement experiments indicate that sequence motifs act in a redundant and combinatorial manner. For explanation of the symbols, see the Figure 3 legend.

FIGURE 6.

Sequence motifs confer regulation to unc-54 3′UTR. For explanation of symbols, see the Figure 3 legend.

FIGURE 7.

Mutational analysis of individual sequence motifs with the three cis-regulatory elements. Note that in constructs 1 and 2 the seed mutations were generated in the context of a mutation in redundantly acting elements because it is only in such a “sensitized” construct that loss of an individual element would have an effect (see Fig. 4). The C deletion in construct 2 (testing the seed match in site A) is not a complete 25-bp deletion, but a CAAAUU motif deletion, which mimics the complete 25-bp deletion. Also note that compared to all other constructs in this article, construct 5 defines the most minimal changes that lead to a complete loss of die-1 3′UTR regulation, namely, the loss of two 6-bp motifs of the same sequence. For explanation of symbols, see the Figure 3 legend.

FIGURE 8.

Differential contribution of the 3′UTR for regulation of expression and activity of the die-1 and cog-1 gene. (A) Schematic representation of the fosmid clones used for expression (panel B) and functional (panel C) analysis of die-1 and cog-1. Fosmid clones are around about 40 kbp in size. Fluorescent reporters were first recombineered into the respective fosmids. In a subsequent recombineering step, 3′UTRs were replaced with the unc-54 3′UTR as schematically indicated. Fosmids were all injected at similar concentration to generate transgenic animals. (B) Testing the effect of 3′UTR replacement on the left/right asymmetric expression of die-1 and cog-1. Representative yfp/mCherry expression of transgenic animals expressing the reporter genes shown in panel A. At least two independent transgenic lines were tested for expression, and expression was found to be almost fully penetrant in that, for the cog-1 reporters, the construct with wild-type 3′UTR was asymmetric while the reporter with the unc-54 3′UTRs was not. For the die-1 reporters, both always showed left/right asymmetric expression regardless of the 3′UTR tested. Bilateral markers (flp-6^prom∷cameleon = ntIs13 and che-1^prom∷mCherry = otIs232) were used to unambiguously identify the ASE neurons (highlighted with stippled lines). Numbers above each panel correspond to the constructs shown in panel A. (C) Testing the effect of 3′UTR replacement on functional derepression of die-1 and cog-1 gene activity. Based on previous work, derepression of die-1 function in ASER is expected to result in a transformation of ASER to ASEL (Johnston et al. 2005), while derepression of cog-1 function in ASEL is expected to result in a transformation of ASEL to ASER (Chang et al. 2003; Johnston et al. 2005; Sarin et al. 2009). Transgenic animals containing reporters with wild-type 3′UTRs or replaced 3′UTR (as shown in panel A) were assayed for their ability to convert cell fate, as assessed with the ASER fate marker gcy-5∷gfp (ntIs1), which is expressed in ASEL if ASER fate is induced (left table) and repressed in ASER if ASEL fate is induced (right table). Each row represents an independent transgenic line. For die-1, we also performed the same experiment with fosmids in which the locus was not tagged with a fluorescent reporter. In that case zero of four lines with the wild-type 3′UTR induced ASEL fate in ASER, and one of four lines with the replaced 3′UTR induced ASEL in ASER in 25% (n = 89) of animals.

FIGURE 9.

Summary. (A) Redundant and combinatorial regulation by three cis-regulatory motifs from the die-1 3′UTR. (B) Revised regulatory loop controlling ASEL/R fate specification. Note that manipulation of cog-1 or lsy-6 activity results in a complete symmetrization of die-1 expression. We have shown here that die-1 regulation occurs on the level of its 3′UTR but also on a non-3′UTR-dependent mechanism, with both mechanisms being cog-1 dependent.