Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis

Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30 degrees C but not 20 degrees C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G(1), consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.     

Three proteins involved in Caenorhabditis elegans vulval invagination are similar to components of a glycosylation pathway

We have molecularly analyzed three genes, sqv-3, sqv-7, and sqv-8, that are required for wild-type vulval invagination in Caenorhabditis elegans. The predicted SQV-8 protein is similar in sequence to two mammalian β(1,3)-glucuronyltransferases, one of which adds glucuronic acid to protein-linked galactose-β(1,4)-N-acetylglucosamine. SQV-3 is similar to a family of glycosyltransferases that includes vertebrate β(1,4)-galactosyltransferases, which create galactose-β(1,4)-N-acetylglucosamine linkages. One model is therefore that SQV-8 uses a SQV-3 product as a substrate. SQV-7 is similar to members of a family of nucleotide-sugar transporters. The sqv genes therefore are likely to encode components of a conserved glycosylation pathway that assembles a C. elegans carbohydrate moiety, the absence of which perturbs vulval invagination.     

Polarity proteins Bem1 and Cdc24 are components of the filamentous fungal NADPH oxidase complex

Regulated synthesis of reactive oxygen species (ROS) by membrane-bound fungal NADPH oxidases (Nox) plays a key role in fungal morphogenesis, growth, and development. Generation of reactive oxygen species (ROS) by the plant symbiotic fungus, Epichloë festucae, requires functional assembly of a multisubunit complex composed of NoxA, a regulatory component, NoxR, and the small GTPase RacA. However, the mechanism for assembly and activation of this complex at the plasma membrane is unknown. We found by yeast two-hybrid and coimmunoprecipitation assays that E. festucae NoxR interacts with homologs of the yeast polarity proteins, Bem1 and Cdc24, and that the Phox and Bem1 (PB1) protein domains found in these proteins are essential for these interactions. GFP fusions of BemA, Cdc24, and NoxR preferentially localized to actively growing hyphal tips and to septa. These proteins interact with each other in vivo at these same cellular sites as shown by bimolecular fluorescent complementation assays. The PB1 domain of NoxR is essential for localization to the hyphal tip. An E. festucae ΔbemA mutant was defective in hyphal morphogenesis and growth in culture and in planta. The changes in fungal growth in planta resulted in a defective symbiotic interaction phenotype. Our inability to isolate a Δcdc24 mutant suggests this gene is essential. These results demonstrate that BemA and Cdc24 play a critical role in localizing NoxR protein to sites of fungal hyphal morphogenesis and growth. Our findings identify a potential shared ancestral link between the protein machinery required for fungal polarity establishment and the Nox complex controlling cellular differentiation.The NADPH oxidases (Nox) are a widely distributed family of eukaryotic proteins that transfer electrons across biological membranes to catalyze the reduction of molecular oxygen to superoxide (1–3). The multiple Nox isoforms found in eukaryotic cells control various physiological and cellular differentiation processes, including cell proliferation, apoptosis and hormone responses in animals (1, 2), and programmed cell death, hormone signaling and root hair tip growth in plants (4).Fungi have three distinct subfamilies of NADPH oxidase (3, 5). NoxA has the core NADPH oxidase transmembrane and catalytic domains but no additional motifs, whereas NoxB has in addition, an N-terminal extension of ∼40 amino acids that is conserved among fungal species that have this isoform (3, 6). NoxC has a longer N-terminal extension, of 170–250 amino acids, which contains a putative calcium-binding EF-hand motif (3), similar to that found in human Nox5 and the plant Rboh enzymes (3, 5). In Aspergillus nidulans, Podospora anserina and Neurospora crassa, NoxA (Nox1) is required for the development of the sexual fruiting body, indicating that a common function of this isoform is regulation of multicellular development (7–10). NoxB (Nox2) is required for ascospore germination in P. anserina and N. crassa (7, 10). However, in Botrytis cinerea, both NoxA and NoxB are required for formation of the multicellular sclerotial sexual structure, but are dispensable for ascospore germination (11).Fungal NADPH oxidases are also required for cellular growth and differentiation processes associated with plant host infection and colonization. In the symbiotic interaction between the endophytic fungus Epichloë festucae and perennial ryegrass, deletion of noxA, but not noxB, disrupted the highly regulated pattern of growth seen in WT associations (6). In the rice blast pathogen, Magnaporthe oryzae, disruption of either nox1 (noxA) or nox2 (noxB) resulted in loss of plant pathogenicity, due to an inability of the fungus to develop a penetration peg beneath the appressorium (12). Both NoxA and NoxB are important for pathogenicity of B. cinerea, but just NoxB is required for formation of the penetration structure (11).NoxR, a fungal homolog of the phagocytic p67^phox Nox regulator, has been shown to regulate both NoxA and NoxB. In the symbiotic fungus E. festucae, a noxR mutant has a similar disrupted symbiotic interaction phenotype as ΔnoxA (6, 13). In B. cinerea and N. crassa, deletion of noxR (nor-1) resulted in a similar developmental phenotype to the noxA(nox1)/noxB(nox2) double mutant (7, 11). Although NoxA and NoxB have distinct cellular functions, it is not yet known how NoxR regulates these two different Nox isoforms. The N-terminal domain of NoxR is similar to p67^phox, and includes four tetratricopeptide repeat (TPR) motifs required for Rac binding and a putative NADPH oxidase activation domain (3, 13) (Fig. 1A). In contrast, the C terminus of NoxR lacks the protein–protein interaction domains present in p67^phox, including Src Homology 3 (SH3) and a conventional Phox and Bem1 (PB1), for interaction with p47^phox and p40^phox, respectively (1, 14). The absence of these domains in NoxR is consistent with the apparent absence of p47^phox and p40^phox homologs in fungal genome databases (3, 13). However, NoxR does possess a nonconventional PB1 domain in the C terminus of the protein, suggesting that fungi have distinct regulatory components that, upon activation, interact with NoxR to translocate this protein from the cytosol to the plasma membrane to assemble and activate the Nox enzyme complex (3, 13) (Fig. 1A).Open in a separate windowFig. 1.(A) Domain structure of E. festucae NoxR, BemA, Cdc24, and CBS1-containing protein. The tetratricopeptide repeat (TPRs), Nox activation (AD), proline-rich region (PRR), Src homology 3 (SH3), Phox and Bem1 (PB1), phox homology (PX), cystathionine beta-synthase 1 (CBS1), and calponin homology (CH) domains are indicated. (B) Yeast two-hybrid assay of the interactions between PB1 domains of E. festucae NoxR, BemA, Cdc24, and CBS1-containing protein. Yeast strain AH109 was transformed with prey and bait vectors, pGADT7 and pGBKT7, as indicated and plated on to SD medium lacking leucine and tryptophan (Upper, -Leu/-Trp) or lacking leucine, tryptophan, histidine, and adenine (Lower, -Leu/-Trp/-His/-Ade). Growth on the latter indicates an interaction between bait and prey. (C) Summary of interactions between PB1 domains based on results shown in B and Fig. S4A. (D) Yeast two-hybrid assay of interactions between full-length NoxR, BemA, and Cdc24. The plating conditions are the same as in B. (E) COS-7 cells were cotransfected with pEF-BOS-Flag-NoxR and pEF-BOS-Myc, or pEF-BOS-Myc-Cdc24 or pEF-BOS-Myc-BemA, or with pEF-BOS-Flag-BemA and pEF-BOS-Myc-Cdc24. Lysates of the transfected cells (Left) were analyzed by immunoprecipitation (IP) with control IgG (Center) or the anti-Flag monoclonal antibody (Right), followed by immunoblotting (WB) with the anti-Myc (Upper) or anti-Flag (Lower) monoclonal antibodies.The objectives of this study were (i) to identify additional components of the fungal Nox enzyme complex by identifying proteins that interact with the PB1 domain of E. festucae NoxR, (ii) to investigate the subcellular localization of these regulators to identify where the Nox complex is assembled, and (iii) to determine whether these proteins, like NoxA and NoxR, have a role in maintaining the mutualistic symbiotic interaction between E. festucae and its host grass perennial ryegrass.     

Cohesins are required for meiotic DNA breakage and recombination in Schizosaccharomyces pombe

In preparation for the unique segregation of homologs at the first meiotic division, chromosomes undergo dramatic changes. The meiosis-specific sister chromatid cohesins Rec8 and Rec11 of Schizosaccharomyces pombe are recruited around the time of premeiotic replication, and Rec10, a component of meiosis-specific linear elements, is subsequently added. Here we report that Rec10 is essential for meiosis-specific DNA breakage by Rec12 (Spo11 homolog) and for meiotic recombination. DNA breakage and recombination also depend on the Rec8 and Rec11 cohesins, strictly in some genomic intervals but less so in others. Thus, in addition to their previously recognized role in meiotic chromosome segregation, cohesins have a direct role, as do linear element components, in meiotic recombination by enabling double-strand DNA break formation by Rec12. Our results reveal a pathway, whose regulation is significantly different from that in the distantly related yeast Saccharomyces cerevisiae, for meiosis-specific chromosome differentiation and high-frequency recombination.     

Ras mutations are rare in solitary cold and toxic thyroid nodules

OBJECTIVE: Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. DESIGN: To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. MEASUREMENTS: Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. RESULTS: Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. CONCLUSION: Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.     

Pulmonary nodules in early fat embolism syndrome: a case report

The radiologic abnormalities in a patient with mild clinical manifestations of fat embolism are reported. The findings consisted of small nodular opacities, which were shown on computed tomography (CT) scans to be located predominantly in the centrilobular and subpleural regions. The nodules presumably represented alveolar edema or hemorrhage secondary to the fat embolism syndrome.     

Exchanges are not equally able to enhance meiotic chromosome segregation in yeast.

Homologous chromosomes pair, and then migrate to opposite poles of the spindle at meiosis I. In most eukaryotic organisms, reciprocal recombinations (crossovers) between the homologs are critical to the success of this process. Individuals with defects in meiotic recombination typically produce high levels of aneuploid gametes and exhibit low fertility or are sterile. The experiments described here were designed to test whether different crossovers are equally able to contribute to the fidelity of meiotic chromosome segregation in yeast. These experiments were performed with model chromosomes with which it was possible to control and measure the distributions of meiotic crossovers in wild-type cells. Physical and genetic approaches were used to map crossover positions on model chromosomes and to correlate crossover position with meiotic segregation behavior. The results show that crossovers at different chromosomal positions have different abilities to enhance the fidelity of meiotic segregation.     

DNA repair systems in early and persistent hepatocyte nodules in the rat

Summary The repair of three DNA lesions, namelyO ⁶-methylguanine, 7-methylguanine, and 3-methyladenine, was investigated within early and persistent hepatocyte nodules generated in Fischer 344 rats by a modified Solt-Farber procedure (diethylnitrosamine initiation followed by a 2-acetylaminofluorene/CCl₄ cycle). TheO ⁶-methylguanine-DNA methyltransferase concentration within both hepatocyte nodule types was always higher than that found in age-matched controls (normal, initiated-only and promoted-only livers). As far as 3-methyladenine and 7-methylguanine-DNA glycosylases are concerned, the early hepatocyte nodules showed far higher activities for both enzymes than were found in the controls, whereas in the persistent ones they underwent a significant decrease. In conclusion hepatocyte nodules are endowed with a high DNA repair activity, which is partly adaptive, partly constitutive; along with others, such a defence mechanism could allow transformed cells to resist many cytotoxic drugs.     

Variant-specific surface proteins of Giardia lamblia are zinc-binding proteins.

Giardia lamblia undergoes surface antigenic variation. The variant-specificsurface proteins (VSPs) are a distinct family of cysteine-rich proteins. Characteristically, cysteineresidues occur mostly as CXXC tetrapeptides. Four of the reported five VSPs contain a putative metal-bindingdomain that resembles other metal-binding motifs; the fifth is closely related but lacks an essentialhistidine. Three different native VSPs bound Zn2+. Co2+, Cu2+, and Cd2+ inhibited Zn2+ binding. Analysisof recombinant VSP fusion proteins showed that the putative binding motif bound Zn2+. Surprisingly, peptidefragments from other regions of the VSP contain numerous CXXCXnCXXC motifs that also bound Zn2+. Analysisof deduced amino acid sequences showed well-conserved CXXC spacing in three out of five VSPs, suggestingconservation of structure despite amino acid sequence divergence. The function of VSPs is unknown, butby binding Zn2+ or other metals in the intestine, VSPs may contribute to Zn2+ malnutrition or inhibitionof metal-dependent intestinal enzymes, which would lead to malabsorption, a well-known consequence ofgiardiasis.     

Megakaryocyte dense granule components are sorted in multivesicular bodies

Recent studies suggest that multivesicular bodies are an intermediate stage in the formation of alpha-granules. In contrast, the kinetics and mode of appearance of dense granules during megakaryocytic maturation has remained poorly understood. Immunoelectron microscopy was used to monitor the appearance of dense granular markers (granulophysin and serotonin) on cryosections of human megakaryocytes (MKs) cultured from CD34(+) precursors. The monitoring was done on days 8 and 13 of culture. The data suggest that dense granules appear in immature MKs early during their maturation, concomitantly with alpha-granule formation. In MKs of intermediary maturation stage, granulophysin was mainly localized within dense granules and multivesicular bodies (MVBs), which were also labeled for serotonin. This study provides evidence that granulophysin is a dense granule marker in human MKs and that MVBs are an intermediary stage of dense granule maturation and probably constitute a sorting compartment between alpha-granules and dense granules. (Blood. 2000;95:4004-4007)     

Minor spliceosome components are predominantly localized in the nucleus

Recently, it has been reported that there is a differential subcellular distribution of components of the minor U12-dependent and major U2-dependent spliceosome, and further that the minor spliceosome functions in the cytoplasm. To study the subcellular localization of the snRNA components of both the major and minor spliceosomes, we performed in situ hybridizations with mouse tissues and human cells. In both cases, all spliceosomal snRNAs were nearly exclusively detected in the nucleus, and the minor U11 and U12 snRNAs were further shown to colocalize with U4 and U2, respectively, in human cells. Additionally, we examined the distribution of several spliceosomal snRNAs and proteins in nuclear and cytoplasmic fractions isolated from human cells. These studies revealed an identical subcellular distribution of components of both the U12- and U2-dependent spliceosomes. Thus, our data, combined with several earlier publications, establish that, like the major spliceosome, components of the U12-dependent spliceosome are localized predominantly in the nucleus.     

Small nodules which could indicate the early phase of autoimmune pancreatitis

We present a case with small pancreatic nodules, which could indicate the early phase of autoimmune pancreatitis (AIP). A 68-year-old man was referred to our hospital for further diagnostic evaluation of a pancreatic mass detected on abdominal ultrasonography screening for epigastric discomfort. Abdominal ultrasonography and endoscopic ultrasonography revealed a low echoic lesion measuring approximately 1 cm with an irregular margin in the body of the pancreas. Computed tomography revealed a tumor in the portal venous phase of enhancement; hence, a distal pancreatectomy was performed. On histology, a marked lymphocyte- and plasma cell-dominant inflammatory cell infiltrate was observed in the nodule. There was another smaller nodule consisting of moderate lymphoplasmacytic infiltration in the 2-cm distal portion of the pancreas. Lymphoplasmacytic infiltration was also observed around the main pancreatic duct in the pancreatic stump. In the parenchyma, other than these 3 portions, the normal lobular structure was well preserved. Little storiform fibrosis and obliterative phlebitis were observed in the resected specimen. On immunohistochemical staining, plasma cells showing strong immunoreactivity for immunoglobulin G4 were observed within these two nodules and around the main pancreatic duct at the cut surface. This case could indicate the early phase and multicentricity of AIP.     

肺磨玻璃肺结节CT三维重在早期肺癌及病理类型的鉴别意义

目的分析肺磨玻璃肺结节(GGO)CT三维重建在早期肺癌及其病理类型的鉴别诊断意义。 方法选择2018年6月至2021年7月我院收治的62例GGO为对象,行CT三维重建检查,对比不同病理类型的CT三维重建参数,分析不同CT三维重建参数对GGO肿瘤性质,对比微浸润性腺癌(MIA)、不典型腺瘤样增生(AAH)、浸润性腺癌(IAC)以及原位腺癌(AIS)患者的体积倍增时间(VDT)。 结果62例GGO倾向中良性11例、MIA 34例、AAH 9例、IAC 6例、AIS 2例。肿瘤的体积、直径、CT值分布,CT值比较P<0.05;IAC患者体积、直径、CT值分布、CT值高于良性、AAH、AIS、MIA(P<0.05);MIA体积、直径、CT值分布、CT值高于良性、AAH、AIS(P<0.05);AIS体积、直径、CT值分布、CT值高于良性、AAH(P<0.05);AAH体积、直径、CT值分布、CT值高于良性(P<0.05),不同病理类型的平均CT值比较(P>0.05)。AAH VDT高于MIA、IAC、AIS(P<0.05),AIS VDT高于IAC、MIA,MIA VDT高于IAC(P<0.05)。 结论GGO体积、直径、CT值分布、CT值,CT三维重建参数在早期肺癌及其病理类型的鉴别诊断中具有临床意义。     

Smoking is a strong risk factor for rheumatoid nodules in early rheumatoid arthritis

OBJECTIVE: To examine whether smoking is a risk factor for rheumatoid nodules in early rheumatoid arthritis, and if so to determine the quantitative effect of smoking. METHODS: From a cohort (n = 1589) in a structured programme for follow up of newly diagnosed cases of rheumatoid arthritis (symptoms of swollen joints < or =12 months), 112 individuals with rheumatoid nodules at inclusion were identified. Nodular patients were each compared with two age and sex matched controls without nodules from the same cohort. A detailed self administered tobacco use questionnaire was answered by 210 patients (63%). RESULTS: Seventy patients were current smokers, 71 former smokers, and 69 had never smoked. Current smoking and former smoking were more common in patients with rheumatoid nodules compared with controls (86% v 59%) in both sexes. Positive rheumatoid factor (RF) was found more often among cases with nodules than controls (78% v 64%). Using detailed information from the questionnaires with conditional logistic regression analyses, ever having smoked was associated with an increased risk of the presence of rheumatoid nodules (odds ratio (OR) = 7.3 (95% confidence interval, 2.3 to 23.6); p = 0.001). The risk of having nodules was not obviously dose dependent when smoking duration as well as smoking amount were examined. A stratified analysis showed that only RF positive smokers had an increased risk of rheumatoid nodules. Smoking was associated with rheumatoid nodules among both men (p = 0.006) and women (p = 0.001). Tobacco use other than smoking (n = 31) was not associated with an increased risk of nodules (OR = 0.8 (0.2 to 3.4); p = 0.813). CONCLUSIONS: There is a strong association between smoking and rheumatoid nodules in early seropositive rheumatoid arthritis.     

Interferon-alpha-inducible proteins are novel autoantigens in murine lupus

OBJECTIVE: To investigate the spectrum of B cell autoimmunity in the recently described anti-CD1-autoreactive T cell receptor (TCR)-transgenic murine lupus-like (CD1 lupus-like) model. METHODS: Lethally irradiated BALB/c/nu/nu mice were injected intravenously with donor BALB/c bone marrow and spleen cells expressing TCRalpha and TCRbeta transgenes that recognize CD1d. Sera from adoptive host animals that developed lupus (i.e., CD1 lupus mice) were collected at serial time points and analyzed by Western blotting and immunoprecipitation, using protein extracts prepared from NIH3T3 mouse fibroblasts and EL-4 lymphocytes, respectively. Sera obtained from older animals in several models of spontaneous lupus (NZB/NZW, MRL++, and MRL/lpr mice), unmanipulated BALB/c/nu/nu mice, and normal BALB/c mice were used as controls. RESULTS: Analyses demonstrated that the prominent targets of autoantibodies in the CD1 lupus-like model are interferon-alpha (IFNalpha)-inducible antigens. Biochemical and serologic characterizations identified one antigen as belonging to the interferon-inducible 202 (Ifi202) subfamily of proteins within the Ifi200 family, and a second antigen as a member of the 70-kd heat-shock protein family. Autoantibodies directed against these antigens were rapidly produced at an early stage of disease. Anti-p50 autoantibodies were present in sera from 7 (78%) of 9 CD1 lupus mice that developed severe kidney disease. CONCLUSION: IFNalpha-inducible proteins represent a novel class of autoantigens in murine lupus, and the findings suggest additional roles for IFNalpha in this disease. Since Ifi202 autoantigens are encoded by the murine non-major histocompatibility complex lupus-susceptibility gene locus Ifi202, these data provide a link between recent advances in lupus genetics and the formation of autoantibodies.     

Phosphoproteins are components of mitotic microtubule organizing centers.

Protein phosphorylation has been suggested as an important control mechanism for the events leading toward the initiation and completion of mitosis. Using a monoclonal antibody recognizing a class of phosphoproteins abundant in mitotic cells, we demonstrated the localization of a subset of these phosphoproteins to several discrete mitotic structures. Patchy immunofluorescence was present in the interphase nuclei, but a significant increase in nuclear immunofluorescence was apparent at prophase. Subsequent mitotic stages demonstrated that immunoreactive material was particularly apparent at microtubule organizing centers, namely, centrosomes, kinetochores, and midbodies. Intense centrosomal localization occurred at the prophase-prometaphase transition and persisted until the reformation of the nuclear membrane in early G1. The cytoplasm of mitotic cells also contained immunoreactive material in sharp contrast to interphase cells that exhibited no cytoplasmic fluorescent staining. Much of the diffuse immunofluorescent cytoplasmic material was removed by a brief lysis of the cells with 0.15% Triton X-100 prior to fixation. The localization of the remaining immunoreactive material after detergent lysis to mitotic microtubule organizing centers suggests that they contain phosphoprotein structural components important, perhaps, in the mitotic phase-interphase transition.     

Centromere-encoded RNAs are integral components of the maize kinetochore

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.