In vivo suppression in yeast strains with plasmids encoding hybrid premRNAs/pretRNAs. (A) The PJ17-1A strain (met4-1_o, lys2-1_o, ade2-1_o), carrying the following hybrid precursors containing the NcoI site (unless otherwise stated), with or without the BHB motif, was patched (two transformants of each construct) on plates lacking methionine: STE2 premRNA/BHB-SUP4 pretRNA, containing the StyI site (patches 1); STE2 premRNA/BHB-SUP4 pretRNA (patches 2); STE2 premRNA/SUP4 pretRNA (patches 3); STE3 premRNA/BHB-SUP4 pretRNA (patches 4); STE3 premRNA/SUP4 pretRNA (patches 5); vector PYX 212 alone (patches V). (B and C) Transformants of the strain GDS4–16D (tap1-1), carrying the hybrid precursor STE2 premRNA/BHB-SUP4 pretRNA containing the StyI site (streak 1), were streaked on plates lacking lysine or adenine (B), or on rich YPD plates (C), compared with the same strain carrying the vector alone (streak V). Red colonies result from a nonsense mutation in the ade2 gene, whereas suppression of this mutation restores the white color.

RT-PCR analysis of the processing of hybrid premRNAs/pretRNAs. RNA was extracted from yeast cells containing different plasmids, reverse-transcribed, amplified, and electrophoresed on an agarose gel, together with size markers (M). STE2 constructs, containing the StyI site (lanes 1 and 2) or NcoI (lanes 3–6), are shown in A, STE3 constructs in B. Hybrid precursors with the BHB motif (A, lanes 1–4; B, lanes 1 and 2) or without the BHB motif (A, lanes 5 and 6; B, lanes 3 and 4) were amplified by using appropriate primers (see Table S1). In A (lanes 2, 4, and 6) and in B (lanes 2 and 4), the amplified DNA was cleaved with SmaI and then reamplified. The DNA bands indicated by an arrow were excised and sequenced. The chromatograms for the spliced products are shown under the respective agarose gels. The two NcoI sites (CCATGG), corresponding to the sequence at the bottom of the inserted stem shown in Fig. 1, are underlined. Arrows above the sequences indicate splice junctions.

Hybrid premRNAs/pretRNAs. The hybrid precursors consist of the receptor premRNA (STE2 or STE3) and the SUP4 pretRNA^Tyr, containing the BHB structure (Upper Left) or without it (Lower Left). The first nucleotide at the 5′-side of the bottom stem of both precursors is position no. 705 in the STE2 premRNA and position no. 594 in the STE3 premRNA. Note that the 5′-side is shown on the right and the 3′-end on the left. Two stop-codons (UAA) are indicated in bold. Upon cleavage of the precursors by the tRNA splicing endonuclease and RNase P (short arrows), and after RNA ligation, two RNA molecules are produced: the mature SUP4 tRNA^Tyr (Middle Right; the anticodon is in bold) and receptor mRNAs containing a stem-loop insertion (Top and Bottom Right). Encoded amino acids in the stem-loop are shown next to the middle codon-base. Arrows indicate the junction site. The bottom part of the stem corresponds to the NcoI site (CCATGG). In one particular hybrid precursor of the STE2 mRNA, the endogenous StyI site (CCTTGG) of the STE2 gene (position no. 705–710) was used: in this case, the nucleotide sequence at the bottom of the stem (right strand) is 5′-CCUUGGUGCGAGAGGA-3′. This sequence is 3 nt longer than the one containing the NcoI site but has the same number of complementary nucleotides. The rest of the construct is unchanged. The encoded amino acid sequence resulting after the splicing of the StyI-containing sequence is NH₂-LGARGKDILSHL-COOH.

Induction of the mating signal transduction pathway. β-Gal activity was assayed in permeabilized cells of strain DDS2. Data are in Miller units and represent averages of three samples; error bars correspond to 1 SD. (A) STE2 gene constructs: STE2 premRNA/pretRNA with the BHB motif, containing the StyI site (column 1); STE2 premRNA/pretRNA with the BHB motif, containing the NcoI site (column 2); STE2 premRNA/pretRNA without the BHB motif, containing the NcoI site (column 3); vector alone (column 4); wild-type STE2 mRNA (column 5). The DDS2 strain is Matα and therefore secretes α-factor, necessary to activate the Ste2 receptor. (B) STE3 gene constructs: STE3 premRNA/pretRNA with the BHB motif (column 1); STE3 premRNA/pretRNA without the BHB motif (column 2); vector alone (column 3); wild-type STE3 mRNA (column 4). The DDS2 strain was incubated together with the strain SG2 (Mata, ste3Δ) as a source of a-factor, necessary to activate the Ste3 receptor.

Chimeric mRNAs can be generated by trans-splicing of pretRNA sequences joined to two different premRNAs. (A) Scheme of the trans-splicing reaction. Splicing sites are those determined by the tRNA domain formed by the two complementary tRNA halves joined to premRNAs 1 and 2. (B) Trans-splicing of STE3 and STE2 premRNAs. The STE3 mRNA 5′-half, joined to the SUP4 tRNA 3′-half, associates with the STE2 mRNA 3′-half, joined to the SUP4 tRNA 5′-half, and reconstitutes the mature tRNA domain, including the BHB motif. Each component is encoded on a different PYX vector. Upon splicing (long arrow) at the tRNA-splice sites, a chimeric mRNA (5′-STE3-STE2-3′) is produced, containing a stem–loop insertion. Short arrows indicate splice sites or junction. (C) RNA of yeast cells containing both plasmids was reverse-transcribed and amplified with appropriate primers (see Table S1). The DNA was electrophoresed on an agarose gel (lane 1) together with size markers (M); the DNA band indicated by an arrow was excised and purified. (D) The chromatogram of the sequence of the DNA purified from the gel is shown. The two NcoI sites (CCATGG), corresponding to the sequence at the bottom of the inserted stem, are underlined; the sequence between the two sites corresponds to the inserted stem–loop. The left sequence of the chromatogram matches STE3, and that on the right matches STE2. The short arrow indicates the splice junction.