Centromere-encoded RNAs are integral components of the maize kinetochore - PubMed (original) (raw)

Centromere-encoded RNAs are integral components of the maize kinetochore

Christopher N Topp et al. Proc Natl Acad Sci U S A. 2004.

Abstract

RNA is involved in a variety of chromatin modification events, ranging from large-scale structural rearrangements to subtle local affects. Here, we extend the evidence for RNA-chromatin interactions to the centromere core. The data indicate that maize centromeric retrotransposons (CRMs) and satellite repeats (CentC) are not only transcribed, but that nearly half of the CRM and CentC RNA is tightly bound to centromeric histone H3 (CENH3), a key inner kinetochore protein. RNAs from another tandem repeat (180-bp knob sequence) or an abundant euchromatic retroelement (Opie) are undetectable within the same anti-CENH3 immune complexes. Both sense and antisense strands of CRM and CentC, but not small interfering RNAs homologous to either repeat, were found to coimmunoprecipitate with CENH3. The bulk of the immunoprecipitated RNA ranged in size from 40 to 200 nt. These data provide evidence for a pool of protected, single-stranded centromeric RNA within the centromere/kinetochore complex.

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Figures

Fig. 2.

Fig. 2.

Centromere-encoded RNAs are coimmunoprecipitated with CENH3. (A) Chromatin samples were immunoprecipitated with anti-CENH3 antibodies, blotted for RNA, and probed with DNA probes for the sequences indicated. Supernatant (S) and pellet (P) fractions for the preimmune and anti-CENH3 treatments are shown. As shown in the bottom lane, RNaseA treatment removed the majority of RNA hybridization. These slot-blot images were used to acquire the numerical data in Table 1, experiment 1. (B) The RNA and DNA %IPs from five different experiments. The RNA %IP data (from Table 1) and associated DNA %IP values from each of five experiments are shown as mean ± SE. (C) High ionic strengths have a minimal impact on the immunoprecipitation of CentC RNA. The preimmmune (S and P) fractions are shown in the top two lanes, standard ChIP (using a 0.3 M NaCl wash) is shown in the middle two lanes, and standard ChIP after a 1 M NaCl wash is shown in the bottom two lanes. This experiment was not repeated in kind, but a second experiment with 0.7 M NaCl gave similar results. (D) Both strands of CRM and CentC are present after anti-CENH3-mediated ChIP. P fractions were blotted for the presence of DNA and RNA, either with or without prior RNaseA treatment. The sense and antisense strands are defined in Methods. This experiment was repeated three times with essentially identical results.

Fig. 3.

Fig. 3.

Centromeric siRNAs are not detected after CENH3-mediated ChIP. (A) The bulk of immunoprecipitated centromeric RNA ranges from 40 to 250 nt in length. The supernatant (S) and pellet (P) fractions are shown for both CRM and CentC. For the P fraction, hybridization with the opposite strand is shown. Hybridization to a 28-nt single-stranded synthetic RNA (SSRNA) homologous to the sense strand of the CRM GAG domain is shown at left. The 28-nt marker is underexposed relative to the other lanes. DNA markers (not shown, but indicated in nt) were used for higher molecular mass estimates. (B) Fragments (≈900 bp) of the CRM GAG domain were detected by strand-specific RT-PCR. This technique is not quantitative. The absence of product when no reverse transcriptase (RT–) was added indicates that the bands were derived from immunoprecipitated RNA, not DNA.

Fig. 1.

Fig. 1.

CRM is actively transcribed. (Upper) A map of CRM element (ref. , later called CRM2; ref. 31). The approximate locations of the GAG, reverse transcriptase (RT), and integrase (INT) domains are shown. (Lower) An agarose gel showing that several internal regions of CRM2 are readily amplified by RT-PCR. The locations of the amplified regions are indicated on the map.

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