TRAMP-mediated RNA surveillance prevents spurious entry of RNAs into the Schizosaccharomyces pombe siRNA pathway - PubMed (original) (raw)

. 2008 Oct;15(10):1015-23.

doi: 10.1038/nsmb.1481. Epub 2008 Sep 7.

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TRAMP-mediated RNA surveillance prevents spurious entry of RNAs into the Schizosaccharomyces pombe siRNA pathway

Marc Bühler et al. Nat Struct Mol Biol. 2008 Oct.

Abstract

In the fission yeast Schizosaccharomyces pombe, the RNA interference (RNAi) machinery is required to generate small interfering RNAs (siRNAs) that mediate heterochromatic gene silencing. Efficient silencing also requires the TRAMP complex, which contains the noncanonical Cid14 poly(A) polymerase and targets aberrant RNAs for degradation. Here we use high-throughput sequencing to analyze Argonaute-associated small RNAs (sRNAs) in both the presence and absence of Cid14. Most sRNAs in fission yeast start with a 5' uracil, and we argue these are loaded most efficiently into Argonaute. In wild-type cells most sRNAs match to repeated regions of the genome, whereas in cid14Delta cells the sRNA profile changes to include major new classes of sRNAs originating from ribosomal RNAs and a tRNA. Thus, Cid14 prevents certain abundant RNAs from becoming substrates for the RNAi machinery, thereby freeing the RNAi machinery to act on its proper targets.

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Figures

Figure 1

Figure 1

Profiling of Ago1-associated small RNAs from wild-type cells. (a) Ago1-associated RNA was isolated and 20–30-nt RNAs were PAGE purified. Small RNA libraries suitable for 454 deep sequencing were generated as described previously. IP, immunoprecipitation. (b) Size distribution and the 5′-most nucleotide of Ago1-associated small RNAs. (c) Classification of Ago1-associated small RNAs isolated from wild-type cells into mitochondrial, repeat-associated, gene-associated, rRNA-associated, tRNA-associated and snoRNA-associated small RNAs. If possible, the orientation of the small RNA with respect to its target is indicated. NA, not applicable. (d) Pie chart illustrating percentages for the individual small RNA classes relative to the total number of small RNAs sequenced from wild-type cells.

Figure 2

Figure 2

Distribution of reads mapping to genomic loci. (a) Distribution of siRNAs at a ura4+ transgene inserted into the outermost centromeric repeats on the right arm of chromosome 1 (otr1R ::ura4+). ura4+ small RNAs show a five-fold preference for the sense strand, and only one of the two strands is found in Ago1. Peaks indicate the number of ura4+ reads with 5′ ends at each genomic position. (b) Zoomed in version of a. Note that nearly all of the reads start with a T (U). (c) Distribution of siRNAs at a centromeric dg repeat. For any given position, generally only one of the two centromeric siRNA strands, starting with a T (U), is present in Ago1. (d) Distribution of sRNAs at the endogenous adh1+ gene.

Figure 3

Figure 3

Profiling of Ago1-associated small RNAs from _cid14_Δ cells. Small RNA libraries suitable for 454 deep sequencing were generated as for wild-type cells. (a) Classification of Ago1-associated small RNAs isolated from _cid14_Δ cells into the same classes as shown in Figure 1. (b) Size distribution and indication of the 5′-most nucleotide of small RNAs. (c) Pie chart illustrating percentages for the individual small RNA classes relative to the total amount of small RNAs sequenced from _cid14_Δ cells. (d) Chromosomal distribution profiles of Ago1-associated small RNAs isolated from wild-type (blue) and _cid14_Δ (red) cells. Blue bullets indicate the location of tRNA genes.

Figure 4

Figure 4

Small RNAs generated from centromeres in wild-type and _cid14_Δ cells. (a) siRNA distribution at centromeres in wild-type (blue) and _cid14_Δ (red) cells. IRC3-L/R, unique inverted repeats flanking both the left and right sides of centromere 3 (ref. 9); blue bullets, tRNA genes in single letter amino acid code. Three identical tRNA-Glu genes are found in centromeric heterochromatin, as well as three noncentromeric genes with identical sequence. Because all reads come from regions of perfect identity, it is ambiguous from which tRNA-Glu locus or loci these reads originate. (b) Quantitative RT-PCR was performed to determine IRC transcript levels in various mutant backgrounds as indicated on the _x_-axes. H3K9me2, dimethylated H3K9. (c) ChIP experiment showing that H3K9me2 in _cid14_Δ cells, where siRNAs are absent, is not affected at IRC3R. DNA from ChIP reactions with or without an antibody against H3K9me2 was used for PCR with primers to amplify the indicated sequences. Error bars are s.d. (d) Cloverleaf schematic of tRNA-Glu. Bold line represents the most prevalent Ago1-associated small RNA (5′-TCCGTCATGGTCCAGTGGCTAGG-3′), which matches the tRNA-Glu 5′ end and D-loop. (e) Northern blot of Ago1-associated RNAs demonstrating that the tRNA-Glu sRNA (indicated with an asterisk) was specifically detected from _cid14_Δ cells, but not from wild-type cells, in a _dcr1_- and _rdp1_-independent manner. (f) Larger tRNA fragments are background contaminating RNAs, because they were also recovered from an untagged Ago1 strain. (g) ChIP experiment showing that H3K9me2 around the tRNA-Glu genes found in centromere 1 is not different in wild-type and _cid14_Δ cells. DNA from ChIP reactions with or without an antibody against H3K9me2 was used for PCR with primers to amplify imr fragments 1–5.

Figure 5

Figure 5

Ribosomal RNAs give rise to antisense siRNAs (rr-siRNAs) in _cid14_Δ cells. (a) Structure of the S. pombe rDNA unit. The long precursor RNA indicated by the arrow is rapidly processed to form the mature 18S, 5.8S and 28S rRNAs through removal of the 5′ and 3′ external transcribed spacers (ETS) and the internal transcribed spacers (ITS) 1 and 2. The nontranscribed spacer (NTS) separates the different rRNA units at the rDNA locus. (b) Antisense rr-siRNAs are produced only in _cid14_Δ cells. Antisense rr-siRNAs are more or less equally distributed along the 18S and 5.8S rRNAs, whereas most of the antisense 28S rr-siRNAs map to the 3′ end. (c) Antisense rr-siRNA biogenesis strictly depends on Rdp1 and Dcr1, but not Clr4. Northern blot was performed with Ago1-associated RNAs isolated from different genetic backgrounds as indicated. The same blot was consecutively hybridized with probes specific for either centromeric dg/dh repeat–associated siRNAs (ra-siRNAs), antisense rr-siRNAs or sense rr-sRNAs.

Figure 6

Figure 6

Model for competition between the RNAi and the Cid14–TRAMP RNA surveillance pathways. In wild-type cells, RDRC and Dicer are recruited to centromeric repeats by the RITS complex, which is tethered to chromatin via siRNA-dependent base-pairing interactions with noncoding centromeric RNA (cenRNA) and association with H3K9 methylated nucleosomes (red lollipops). This results in dsRNA synthesis and the generation of repeat-associated siRNAs (rasiRNAs), which mediate further RITS recruitment coupled to H3K9 methylation by the Clr4-containing CLRC methyltransferase complex. The TRAMP complex targets rRNA fragments for exosomal degradation. In _cid14_Δ cells, rRNA fragments accumulate and become substrates for RDRC and Dicer. This titrates RDRC and Dicer away from cenRNA, resulting in the generation of rRNA-siRNAs (rr-siRNAs) and a reduction in rasiRNAs.

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