Introns are necessary for mRNA accumulation in Schizophyllum commune

Abstract

The cDNA coding sequence of the Agaricus bisporus hydrophobin gene ABH1 under the regulation sequences of the Schizophyllum commune SC3 hydrophobin gene gave no expression in S. commune. In contrast, the genomic coding sequence (containing three introns) produced high levels of ABH1 mRNA when transformed to S. commune in the same configuration. Apparently, introns were needed for the accumulation of mRNAs from the ABH1 gene. When the effect of intron deletion on expression of the homologous genes SC3 and SC6 was examined, it was observed that only the genomic coding sequences were expressed in S. commune. Run-on analysis with nuclei harbouring intron-containing and intronless SC6 showed that this effect did not occur at the level of transcription initiation: genomic and cDNA sequences were equally active in this respect. When a 50 bp artificial intron containing the consensus splice and branch sites of S. commune introns, in addition to random-generated sequences, was introduced in the right orientation into the intronless SC3 transcriptional unit, accumulation of SC3 mRNA was restored. By polymerase chain reaction amplification, no unspliced SC3 mRNA species could be detected. Furthermore, the addition of an intron into the transcriptional unit of the gene for green fluorescent protein (GFP) effected clear fluorescence of the transgenic hyphae. Apparently, splicing is required for the normal processing of primary transcripts in S. commune.

Polarized Gene Expression Determines Woronin Body Formation at the Leading Edge of the Fungal Colony

“…We also demonstrate an important role for intron splicing in Neurospora, and similar findings in the filamentous basidiomycete Schizophyllum commune (Lugones et al, 1999) suggest that coupling between mRNA splicing and gene expression may be common in filamentous fungi. Intron splicing is required for the accumulation of hex-1 transcripts, and this effect is probably achieved at the posttranscriptional level (Supplemental Figure 1), suggesting that, as in metazoan systems (Maniatis and Reed, 2002), intron splicing is coupled to mRNA transport or stability.…”

Elevation of Intracellular cAMP Levels by Dominant Active Heterotrimeric G Protein Alpha SubunitsScGP-AandScGP-Cin Homobasidiomycete,Schizophyllum commune

“…The expression of GFP (S65T) in S. commune has been reported. 28) The expression of EGFP has also been confirmed in S. commune (T. Kinoshita et al, personal communication). To screen the clones strongly expressing EGFP, the fluorescent intensity of each clone was measured by fluorometer (Fig.…”

“…The NcoI site contained the first Met, and the XbaI site was located at the rear of the EGFP stop codon. Since it has been reported that introns are necessary for normal processing of GFP in S. commune, 28) the GPD (glyceraldehyde-3-phosphate dehydrogenase) gene of S. commune 29) was used to stabilize EGFP primary transcripts. Primers GPD-Nco-F and GPD-Nco-R were used to amplify the genomic fragment covering the full region of the GPD gene (from the first Met to 337 leucine adjacent to the stop codon).…”

Functional expression of the green fluorescent protein in the ectomycorrhizal model fungus Hebeloma cylindrosporum

“…Recently, Burns et al (2005) successfully transformed the mushroom Agaricus bisporus with a GFP expressing vector and concluded that efficient GFP expression in A. bisporus requires introns. Introns were also used for GFP expression in the two previously transformed homobasidiomycetes Schizophyllum commune (Lugones et al 1999) and Phanerochaete chrysosporium (Ma et al 2001). Nevertheless, Rodriguez-Tovar et al (2005) recently reported GFP expression in Pisolithus tinctorius without the requirement of introns.…”