Eukaryotic genomes are littered with intronic sequences that must be removed for proper mRNA processing. Curiously, genes encoding developmental regulators often have large first introns1. One such regulator is C. elegans
mpk-1, which encodes ERK/MAP kinase. The
mpk-1 locus encodes two isoforms-
mpk-1b has a unique first exon followed by an 8.2 kb intron, while
mpk-1a and
mpk-1b share all other exons and introns. Germline MPK-1 is required for fertility2 with
mpk-1b strongly expressed3. We assayed MPK-1 protein expression with epitope tags in both the
mpk-1b-specific exon and the shared C terminus, and assayed MPK-1B function with a frameshift that causes a premature stop codon in the
mpk-1b specific exon. The pattern of MPK-1B-specific staining matched staining of the C-terminal MPK-1AB tag and
mpk-1b frameshift mutants were sterile, consistent with MPK-1B being the primary germline isoform. We visualized
mpk-1 RNAs in the germline using single molecule fluorescence in situ hybridization (smFISH) and quantified transcripts localized in the nucleus to active transcription sites (ATS) using MATLAB. We compared ATS signals from two distinct probes: one specific to the 8.2 kb first intron and one specific to exons. We expected to see confirmation of
mpk-1b ATS by nuclear intron/exon overlap (IEO); however, the two probes generated distinct ATS patterns with only about 30% IEO in the ATS of germline stem cells (GSC) and nearly 100% IEO in pachytene ATS. Remarkably, two other genes with large introns,
lag-1 and
lag-3/sel-8, had a similarly graded ATS pattern. A 1 kb deletion in the middle of the
mpk-1b intron causes temperature-sensitive sterility and a smaller than normal progenitor zone. Moreover, preliminary data suggest that the IEO in GSCs increases from 30% to 70% in this intron mutant. We propose that large introns either slow transcription or are processed in a regulated fashion to modulate gene expression. 1Bradnam & Korf (2008) PLoS ONE, 2Church et al. (1995) Development, 3Lee et al. (2007) PLoS Genetics