Figure 1. Identification of a minimal fragment of LST-1 that interacts with FBF-2.

(A) Yeast 2-hybrid analyses of interaction between the FBF-2 PUM domain fused to a GAL4 activation domain (A.D.) and LST-1 fragments fused to the LexA DNA-binding domain (D.B.D.). A negative control empty vector (EV) with no FBF-2 fused to the activation domain and a positive control with the FBF-2 PUM domain fused to the activation domain were assessed with LST-1 34–328 fused to the DNA-binding domain and are shown at the top of the graph. (B) LST-1 L83 is critical for interaction with FBF-2. Yeast 2-hybrid analyses were conducted with LST-1 residues 55–105 fused to a GAL4 activation domain and the PUM domain of FBF-2 fused to the LexA DNA-binding domain. Mutants in LST-1 that interfered with FBF-2 interaction are colored green and those that were competent for interaction are colored gray. Binding activity is shown as units of β-galactosidase (β-gal) activity normalized to cell count. Error bars indicate the standard deviation of three biological replicate measurements. A schematic representation of the yeast 2-hybrid assay is illustrated in Figure 1—figure supplement 1 and results of yeast 2-hybrid analyses of LST-1 and FBF homologs are shown in Figure 1—figure supplement 2.

Figure 1—figure supplement 1. A schematic of the yeast two-hybrid assay.

To quantify binding activity, fusion proteins are introduced into yeast with a ‘bait’ protein (purple) fused to the LexA DNA-binding domain. The LexA protein provides a tether to the promoter region of the β-gal reporter gene by virtue of its association with the LexA operator site. ‘Prey’ proteins (peach) are introduced as fusions to the GAL4 transcriptional activation domain. Transcription of the reporter gene is dependent on interaction between the bait and prey proteins, which recruits RNA polymerase to the reporter gene.

Figure 1—figure supplement 2. LST-1 interacts with FBF but not homologous PUF proteins.

Yeast-two hybrid assays were conducted with LST-1 residues 55–105 fused to the Lex-A DNA-binding domain (D.B.D.) and the GAL4 activation domain (A.D.) was fused to the PUM domain of the indicated PUF protein homologs: FBF-1 (residues 121–614), human PUM1 (residues 456–1064), human PUM2 (residues 456–1064), C. elegans PUF-8 (full length), and D. melanogaster dPUM (residues 1091–1426). Binding activity is shown as units of β-gal activity normalized to cell count. Error bars indicate the standard deviation of three biological replicate measurements.