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. 2003 Aug 1;22(15):3983-91.
doi: 10.1093/emboj/cdg384.

Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes

Francisco E Nicolás  1 , Santiago Torres-Martínez, Rosa M Ruiz-Vázquez
Affiliations

Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes

Francisco E Nicolás et al. EMBO J. .

Abstract

Transformation of Mucor circinelloides with self-replicative plasmids containing a wild-type copy of the carotenogenic gene carB causes silencing of the carB function in 3% of transformants. Genomic analyses revealed a relationship between silenced phenotype and number of copies of plasmids. This phenotype results from a reduction of the steady-state levels of carB mRNA, a reduction that is not due to differences in the level of transcription, indicating that silencing is post-transcriptional. Small sense and antisense RNAs have been found to be associated with gene silencing in M. circinelloides. Two size classes of small antisense RNAs, differentially accumulated during the vegetative growth of silenced transformants, have been detected: a long 25-nucleotide RNA and a short 21-nucleotide RNA. Secondary sense and antisense RNAs corresponding to sequences of the endogenous gene downstream of the initial triggering molecule have also been detected, revealing the existence of spreading of RNA targeting in fungi. These findings, together with the self-replicative nature of the triggering molecules, make M. circinelloides a suitable organism for investigating some unresolved questions in RNA silencing.

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Figures

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Fig. 1. (A) Schematic representation of the carB genomic region. The transcription start and polyadenylation sites (arrows), the translation start and termination codons, and the exons and introns (open and shaded boxes) are indicated. Above the scheme, carB riboprobes utilized to detect small sense and antisense RNAs. Below, carB constructs used for transformation. The length (in base pairs) of the carB sequence in each construct is indicated. (B) Northern blot analysis of transformants containing carB exogenous sequences. RNAs were extracted from dark-grown or light-pulsed mycelia of five pMAT754 transformants showing different phenotypes. About 5 µg of RNA was loaded in each lane and hybridized with a 1.8 kb cDNA fragment of the carB gene. The membrane was reprobed with a 25S rRNA probe to check loading. Illuminated mycelia were exposed to blue light for 4 min at 4 W/m2, and incubated in the dark for 20 min before RNA isolation. Densitometric analysis was used to estimate the mRNA levels.
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Fig. 2. Southern blot analysis of transformants containing carB exogenous sequences. Total DNA (0.75 µg) from the wild-type strain R7B (WT) and albino and wild-type transformants was digested with EcoRI and XhoI, and hybridized with a 1.8 kb cDNA fragment of carB. (A) Wild-type (T1-T3) and albino (T4-T5) transformants obtained with plasmid pMAT647. (B) Albino (T6-T9) and wild-type (T10-T13) transformants obtained with plasmid pMAT754. The EcoRI–XhoI digestion generates a 3.6 or 3.7 kb fragment from plasmids pMAT754 and pMAT647, respectively, which include the complete carB insert. The 4.5 kb fragment corresponds to the endogenous carB gene. Densitometric analysis of the hybridizing bands was used to calculate the copy number of carB exogenous sequences per nucleus.
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Fig. 3. Analysis of the methylation state of carB exogenous and endogenous sequences in pMAT754 transformants. DNA of the wild-type stain R7B (WT) and albino (T6-T7) and wild-type (T10-T11) transformants was restricted with the isoschizomers MspI (M) and HpaII (H) (insensitive and sensitive to cytosine methylation, respectively), or with the enzyme ClaI, which is sensitive to cytosine methylation. Filter was hybridized with a 2.4 kb PstI–XhoI fragment of plasmid pMAT754, which contains the carB coding and promoter sequences. Arrows indicate fragments of 862 bp (HpaII–MspI digestion) and 888 bp (ClaI digestion), derived solely from endogenous carB sequences. Asterisks mark fragments exclusively derived from carB exogenous sequences. The sizes (in base pairs) of fragments of the 1 kb Plus ladder marker (Gibco-BRL) are indicated.
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Fig. 4. RNase protection experiments for carB transcripts. (A) Schematic representation of endogenous carB transcripts, showing the expected sizes of fragments protected by unspliced and spliced carB mRNA. The rectangle represents the transcribed region of the endogenous carB gene. Shaded box indicates an intron (I) of the carB gene. The RNA probe (277 nt) was generated from plasmid pMAT643 and is indicated by a solid line above the carB gene. The probe contains sequences derived from the plasmid, which are indicated by the diagonal portion of the solid line. (B) RNase protection analysis was performed on total RNA isolated from light-pulsed or dark-grown mycelia of the wild-type strain (w) or two albino pMAT754 transformants (a1 and a2). A pyrG probe (202 nt) generated from plasmid pMAT645 was used as control to normalize the RNA quantity. This probe protects a 158 nt fragment of the pyrG mRNA. Lanes 1–4 correspond to a filter exposed for 1 h, and lanes 5–14 correspond to a filter exposed for 15 h. Lane 1, undigested pyrG probe; lane 2, undigested carB probe; lanes 3–8, RNase protection experiments using both the carB and the pyrG probes on RNA isolated from light-pulse mycelia; lanes 9 and 10, RNase protection experiments using only the carB probe; lanes 11 and 12, RNase protection experiments using both the carB and the pyrG probes on RNA isolated from dark-grown cultures; lane 13, carB probe digested with RNase; lane 14, pyrG probe digested with RNase.
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Fig. 5. Sense and antisense RNAs associated with post-transcriptional gene silencing of the carB gene. Fifteen micrograms of low molecular weight RNAs isolated from unsilenced wild-type strain (wt) or two different silenced albino transformants (a3 and a4) grown for three days under continuous illumination conditions were loaded in each lane. Ten picomoles per lane of 29-mer DNA oligonucleotide in antisense orientation (AS) and 25-mer DNA oligonucleotide in sense orientation (S) were used as size markers and to control the hybridization specificity. (A) RNA blot hybridized with a carB antisense-specific riboprobe (Figure 1A, probe a′). The same filter was re-hybridized with a carB sense-specific probe to detect the position of the 25 nt oligonucleotide (marked by an arrow). (B) Identical RNA blot hybridized with a carB sense-specific riboprobe (Figure 1A, probe a). Equal loading of the small RNA species was confirmed by EtBr staining of the predominant RNA species in the samples after the small RNAs were separated by agarose gel electrophoresis (data not shown).
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Fig. 6. Differential accumulation of 21 and 25 nt antisense RNAs during vegetative growth of silenced strains. Low-molecular weight RNAs were isolated from a silenced albino transformant of plasmid pMAT647 (a3), grown for different times (hours) in liquid culture and from spores collected from solid medium. Fifteen micrograms of RNA was loaded in each lane. Ten picomoles per lane of 29-mer DNA oligonucleotide in antisense orientation (AS) and 25-mer DNA oligonucleotide in sense orientation (S) were used as size markers. (A) RNA blot hybridized with a carB antisense-specific riboprobe (Figure 1A, probe a′). The predominant RNA species in the small RNA samples (sRNA) were stained with EtBr after RNAs were separated by agarose gel electrophoresis. (B) Identical RNA blot hybridized with a carB sense-specific riboprobe (Figure 1A, probe a).
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Fig. 7. Secondary sense and antisense RNAs in transgene-induced gene silencing. Low-molecular weight RNAs were isolated from the wild-type strain (wt), an albino transformant containing a full-length carB transgene (a3; plasmid pMAT647) and an albino transformant containing 3′-truncated carB exogenous sequences (a5; plasmid pMAT650). Cultures were grown for 24 h in liquid medium under continuous illumination conditions. Fifteen micrograms of RNA was loaded in each lane. (A) RNA blot hybridized with a carB antisense-specific riboprobe (Figure 1A, probe a′). (B) Identical RNA blot hybridized with the carB sense-specific riboprobe (Figure 1A, probe a). (C) RNA blot hybridized with the 5′-end carB antisense-specific riboprobe (Figure 1A, probe b′). (D) Identical blot hybridized with the 3′-end carB antisense-specific riboprobe (Figure 1A, probe c′). Ten picomoles per lane of 29-mer (A and B), 25-mer (C) or 30-mer (D) DNA oligonucleotides in antisense orientation (AS) and 25-mer DNA oligonucleotides in sense orientation (S) were used as size markers.
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References

    1. Ausubel F.M., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. (1989) Current Protocols in Molecular Biology. Green Publishing Associates and Wiley-Interscience, New York, NY.
    1. Bartnicki-Garcia S. and Nickerson,W.J. (1962) Induction of yeast-like development in Mucor by carbon dioxide. J. Bacteriol., 84, 829–840. - PMC - PubMed
    1. Benito E.P., Campuzano,V., López-Matas,M.A., De Vicente,J.I. and Eslava,A.P. (1995) Isolation, characterization and transformation, by autonomous replication, of Mucor circinelloides OMPdecase-deficient mutants. Mol. Gen. Genet., 248, 126–135. - PubMed
    1. Bernstein E., Caudy,A.A., Hammond,S.M. and Hannon,G.J. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 409, 363–366. - PubMed
    1. Catalanotto C., Azzalin,G., Macino,G. and Cogoni,C. (2002) Involvement of small RNAs and role of the qde genes on the gene silencing pathway in Neurospora crassa. Genes Dev., 16, 790–795. - PMC - PubMed

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