Genetics, Vol. 176, 749-761, June 2007, Copyright © 2007
doi:10.1534/genetics.107.071902

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: genetics.107.071902v1 176/2/749    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Neumann, P. Articles by Jiang, J. Search for Related Content PubMed PubMed Citation Articles by Neumann, P. Articles by Jiang, J.

The Centromeric Retrotransposons of Rice Are Transcribed and Differentially Processed by RNA Interference

Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706

¹ Corresponding author: Department of Horticulture, University of Wisconsin, 1575 Linden Dr., Madison, WI 53706.
E-mail: jjiang1{at}wisc.edu

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
Retrotransposons consist of significant portions of many complex eukaryotic genomes and are often enriched in heterochromatin. The centromeric retrotransposon (CR) family in grass species is colonized in the centromeres and highly conserved among species that have been diverged for >50 MY. These unique characteristics have inspired scientists to speculate about the roles of CR elements in organization and function of centromeric chromatin. Here we report that the CRR (CR of rice) elements in rice are highly enriched in chromatin associated with H3K9me2, a hallmark for heterochromatin. CRR elements were transcribed in root, leaf, and panicle tissues, suggesting a constitutive transcription of this retrotransposon family. However, the overall transcription level was low and the CRR transcripts appeared to be derived from relatively few loci. The majority of the CRR transcripts had chimerical structures and contained only partial CRR sequences. We detected small RNAs (smRNAs) cognate to nonautonomous CRR1 (noaCRR1) and CRR1, but not CRR2 elements. This result was also confirmed by in silico analysis of rice smRNA sequences. These results suggest that different CRR subfamilies may play different roles in the RNAi-mediated pathway for formation and maintenance of centromeric heterochromatin.

Phylogenetic analysis showed that the CRR family has diverged into two subfamilies of autonomous elements (CRR1 and CRR2) and two subfamilies of nonautonomous elements (noaCRR1 and noaCRR2) (NAGAKI et al. 2005). Close CRR relatives identified in several distantly related grass species (MILLER et al. 1998), including maize (centromeric retrotransposon of maize, CRM) (ZHONG et al. 2002; NAGAKI et al. 2003), barley (Cereba elements) (PRESTING et al. 1998; HUDAKOVA et al. 2001), and sugarcane (NAGAKI and MURATA 2005), are almost exclusively located in centromeres. These results suggest that this family of retrotransposons colonized in the centromeres of the common ancestor of these grass species and has maintained its centromeric specificity for >50 MY (KELLOGG 2001). The comparison of centromeric retrotransposons from rice, maize, and barley revealed several highly conserved motifs even within their long terminal repeats (LTRs), suggesting that the sequences might have evolved under selective pressure at the DNA level (NAGAKI et al. 2003).

Repetitive DNA elements located in the centromeric heterochromatin are often transcriptionally silent. However, a low level of transcription paradoxically is required for establishing the transcriptionally silent state of heterochromatin through RNA interference (RNAi) (reviewed in BERNSTEIN and ALLIS 2005; MATZKE and BIRCHLER 2005). In the RNAi pathway, double-stranded RNA (dsRNA) derived from repetitive DNA sequences is processed by the RNAi machinery into short interference RNA (siRNA). The siRNAs then participate in RNAi by targeting other RNA molecules for degradation and also by driving heterochromatin formation at complementary genomic sites (GREWAL and RICE 2004; VERDEL et al. 2004; CHAN et al. 2005; GENDREL and COLOT 2005). Thus, the importance of this pathway not only lies in silencing of repetitive DNA elements, which are frequent targets of RNAi, but also seems to be crucial for heterochromatin formation and centromere function, which has been documented in several species, including Schizosacharomyces pombe (VOLPE et al. 2003; CAM et al. 2005; PIDOUX and ALLSHIRE 2005), humans (FUKAGAWA et al. 2004), mice (KANELLOPOULOU et al. 2005), Drosophila (PAL-BHADRA et al. 2004; DESHPANDE et al. 2005), and Trypanosoma brucei (DURAND-DUBIEF and BASTIN 2003).

The high abundance and centromere-specific localization of the CRR elements in rice provide an ideal system for studying the transcription and its association with heterochromatin formation and centromere function of the centromeric repeats. Here we report a comprehensive transcription study of the CRR elements using a combination of bioinformatics and experimental approaches. We demonstrate that CRR transcripts are present in all tested rice organs, suggesting that the transcription of CRR sequences is constitutive. Transcription initiation as well as termination occur both inside and outside of CRR elements. Some of the CRR transcripts are processed into small RNA (smRNA). The potential impact of CRR transcription on centromeric heterochromatin formation and centromere function is discussed.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
Chromatin immunoprecipitation–polymerase chain reaction, reverse transcriptase–polymerase chain reaction, and 3' rapid amplification of cDNA ends analyses:
A japonica rice variety Nipponbare was used in all experiments. Chromatin immunoprecipitation (ChIP) and ChIP–polymerase chain reaction (ChIP–PCR) followed published protocols (YAN et al. 2005). Two sets of primers were designed from the CRR1, CRR2, and noaCRR1 elements. These primers were designed from the LTR of CRR1 (CRR1_LTRF1 and CRR1_LTRR1), CRR2 (CRR2_LTRF1 and CRR2_LTRR1), and noaCRR1 (noaCRR1_LTRF1 and noaCRR1_LTRR1); the gag region of CRR1 (CRR1_CDF1 and CRR1_CDR1) and CRR2 (CRR2_CDF1 and CRR2_CDR1); and the 5'-UTR region of noaCRR1 (noaCRR1_insF1 and noaCRR1_insR1) (supplemental Table 1 at http://www.genetics.org/supplemental/). Reverse transcriptase–polymerase chain reaction (RT–PCR) and 3' rapid amplification of cDNA ends (RACE) were according to LEE et al. (2006). Random hexamers were used for the reverse transcription in RT–PCR, and the 3'-RACE_oligoT primer (5'-GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT TTV-3') was used in 3'-RACE. RT–PCR reaction, using primers specific for actin, actin_F1 (5'-CTC GTC TGC GAT AAT GGA A-3'), and actin_R1 (5'-ATC CTC CAA TCC AGA CAC TG-3'), was carried out as a control of equal RNA loading. GeneRACER kit (Invitrogen, San Diego) was used for 5'-RACE. Nested PCR was employed to increase the sensitivity and specificity of both RACE methods. Products of the first PCR were used as a template in the second PCR that was carried out using nested primers. The first 3'-RACE PCR was done using primers CRR_ppt1F and AUAP_3'-RACE (5'-GGC CAC GCG TCG ACT AGT AC-3'). 3'-RACE nested PCR was done using a primer specific to a particular CRR subfamily (CRR1_1F, CRR2_1F, or noaCRR1_1F) and AUAP_3'-RACE. The first 5'-RACE PCR was done using primers CRR_pbs1F and RACER_PN1 (5'-GGA GCA CGA GGA CAC TGA-3'). The 5'-RACE nested PCR was done using a primer specific for a particular CRR subfamily (CRR1_2R, CRR2_2R or noaCRR1_2R) and RACER_PN2 (5'-CAC TGA CAT GGA CTG AAG GA-3').

Cloning, sequencing, and sequence analyses:
Both RT–PCR and RACE products were cloned into pCRII TOPO vector using the TOPO TA cloning kit (Invitrogen). The sequencing of selected clones was done by the dideoxy-mediated chain-termination method using the BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA). Sequences of RT–PCR and RACE products were assembled using Staden Package software (STADEN 1996) and all of them were submitted into the GenBank database (supplemental Table 2 at http://www.genetics.org/supplemental/).

GenBank EST (BOGUSKI et al. 1993) and KOME full-length cDNA (fl-cDNA) (KIKUCHI et al. 2003) were searched for CRR-similar sequences using the Blast program (ALTSCHUL et al. 1997). The sequences were analyzed using programs implemented at the Biology WorkBench website (http://workbench.sdsc.edu/), EMBOSS (RICE et al. 2000), and Staden Package software (STADEN 1996). Preliminary sequence comparisons were done using the Dotter and J-dotter programs (SONNHAMMER and DURBIN 1995; BRODIE et al. 2004). Multiple alignments were made using CLUSTALW (THOMPSON et al. 1994). RPS-Blast (MARCHLER-BAUER et al. 2003) and InterProScan (ZDOBNOV and APWEILER 2001) were used to search conserved domain and InterPro protein databases, respectively. Splice site analysis was performed using the FSPLICE program implemented at the SoftBerry server (http://www.softberry.com/berry.phtml). Databases of CRR elements, RT–PCR products, fl-cDNAs, ESTs, and rice genome pseudomolecules were screened and compared with one another using Standalone Blast. The database of CRR elements contained all sequences published by NAGAKI et al. (2005). Sequences of the rice genome pseudomolecules (build 4.0 release) were downloaded from the International Rice Genome Sequencing Project website (http://rgp.dna.affrc.go.jp/IRGSP/Build4/build4.html). Sequences of CRR elements that we considered typical for each CRR subfamily and used for database searches and basic sequence comparisons were downloaded from the GenBank database under the following accession nos.: AY827957 (CRR1_CH1-2), AY828150 (CRR1_CH10-1), AY828151 (CRR1_CH10-2), AY827959 (CRR2_CH1-1), AY828019 (CRR2_CH4-2), AY828024 (CRR2_CH4-7), AY828152 (CRR2_CH10-1), AY828039 (noaCRR1_CH4-5), and AY828004 (noaCRR2_CH1-3). Two databases of smRNA sequences were searched to identify CRR-derived smRNA sequences (SUNKAR et al. 2005b; JOHNSON et al. 2007). Sequences of all CRR elements described by NAGAKI et al. (2005) and sequences of RT–PCR and RACE products were used as queries in the smRNA database search.

Northern blot hybridization:
Total RNA was isolated from leaves and treated with DNaseI (Ambion, Austin, TX). Approximately 40 µg RNA was loaded in each lane and resolved on denaturing 1% agarose gel and then transferred on Nytran SPC nylon membrane (Schleicher & Schuell BioScience, Keene, NH). Probes were radioactively labeled with 3000 Ci/mmol [-³²P]dATP (Amersham Biosciences, Piscataway, NJ) using the Strip-EZ DNA kit (Ambion). PCR-amplified inserts from clones ID51 (CRR1 LTR; DV468825), ID69 (CRR2 LTR; DV468838), ID13 (CRR1 reverse transcriptase coding domain, rt; DV468787), ID18 (CRR2 rt; DV468782), ID101 (noaCRR1 LTR; DV468869), and ID45 (noaCRR1 gag; DV468819) were used as templates for the labeling. The hybridization was performed overnight in 125 mM sodium phosphate buffer (pH 7.2) containing 50% deionized formamide, 7% SDS, and 250 mM sodium chloride at 50°. After the hybridization, membranes were washed twice in 2x SSC and 0.1% SDS for 15 min, twice in 0.2x SSC and 0.1% SDS for 15 min, and, finally, once in 5x SSC and 0.5% SDS for 10 min at 65°. Signals were detected using a phosphorimager.

Detection of smRNA:
Low-molecular-weight RNA was isolated using the mirVana microRNA (miRNA) isolation kit (Ambion). Ten micrograms of RNA was resolved on denaturing 15% polyacrylamide gel and then transferred to Nytran SPC nylon membrane (Schleicher & Schuell BioScience). Probes were labeled with 800 Ci/mmol [-³²P]UTP using the MAXIscript kit for in vitro transcription labeling (Ambion). The templates for in vitro transcription were prepared from RACE clones ID51 (CRR1 LTR; DV468825), ID69 (CRR2 LTR; DV468838), and ID101 (noaCRR1 LTR; DV468869). Promoter sequences for T7 polymerase were added to the CRR sequences by PCR using primer pairs T7+CRR1_1F (5'-TAA TAC GAC TCA CTA TAG GGT GAT GAG GAC ATC CCT TCC-3') and AUAP_3'-RACE, T7+CRR2_1F (5'-TAA TAC GAC TCA CTA TAG GGT GAT GAG GAC ATC AAC ACC A-3') and AUAP_3'-RACE or T7+RACER_PN2 (5'-TAA TAC GAC TCA CTA TAG GGC ACT GAC ATG GAC TGA AGG A-3') and noaCRR1_2R. To visualize marker RNA, 0.5 fmol of the marker-specific template was added to the labeling reactions. The hybridization was performed overnight in 125 mM sodium phosphate buffer (pH 7.2) containing 50% deionized formamide, 7% SDS, and 250 mM sodium chloride at 42°. After the hybridization, membranes were washed three times in 2x SSC and 0.1% SDS for 10 min, twice in 1x SSC and 0.1% SDS for 15 min, and, finally, once in 5x SSC and 0.5% SDS for 10 min at 50°. DNA oligonucleotide probes P98-A8 (5'-ACA CGG ACA TCT ACA GAA AAA-3') and OsmiR156 (5'-TGT GCT CAC TCT CTT CTG TCA-3') were labeled at 5'-ends with [-³²P]ATP (Amersham Biosciences) using T4 polynucleotide kinase (Invitrogen) according to manufacturer's protocol. For these two probes, temperatures of hybridization and washing were changed to 38° and 42°, respectively. Signals were detected using a phosphorimager.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
CRR elements are enriched in chromatin associated with histone H3 dimethylation at Lys 4:
Cytological analysis showed that both CRR1 and CRR2 elements are almost exclusively located in the centromeres, whereas noaCRR1 elements are located in both centromeric and pericentromeric regions (NAGAKI et al. 2005). We performed ChIP analysis to reveal whether the CRR elements are preferentially associated with rice heterochromatin. We used antibodies against histone H3 dimethylation at Lys 4 (H3K4me2), which is preferentially associated with transcriptionally active euchromatin, and histone H3 dimethylation at Lys 9 (H3K9me2), which is a hallmark of repressive heterochromatin (JENUWEIN and ALLIS 2001). Two pairs of PCR primers were designed from the CRR1, CRR2, and noaCRR1 elements. Quantitative real-time PCR analysis revealed significant enrichments of the CRR sequences in the immunoprecipitated DNA associated with H3K9me2 but not with H3K4me2 (Figure 1). These results demonstrate that CRR1, CRR2, and noaCRR1 elements are highly enriched in chromosomal domains associated with heterochromatin.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   FIGURE 1.— Association of CRR elements with H3K4me2 and H3K9me2. ChIP–PCR primers were designed from the LTR (CRR1, CRR2, noaCRR1), gag region (CRR1 and CRR2), and 5'-UTR (noaCRR1). The presence of H3K4me2 or H3K9me2 was inferred when enrichment of an antibody-binding fraction over mock DNA was significant in Student's t-test ( = 0.05, n = 3). Relative fold enrichment (RFE) is calculated as described (YAN et al. 2005) and indicated by the height of the bar with standard error; the dashed line (RFE = 1) represents the RFE of the reference gene Cen8.t00238 that shows no H3K4me2 and no H3K9me2 (YAN et al. 2005). C1 (gene Cen8.t00390; see YAN et al. 2005) is a positive control for H3K4me2. C2 (gene Cen8.t00633; see YAN et al. 2005) is a positive control for H3K9me2.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   FIGURE 2.— Structure of CRR-related fl-cDNAs and their corresponding genomic loci. The fl-cDNA sequences (exons) are shown as white rectangles and their orientation is from the 5'- to the 3'-end. The only exception is fl-cDNA AK069963 (chromosome 11), which is shown in the opposite orientation. The longest ORFs are highlighted in blue. The genomic loci are named A–S and their positions in the rice chromosome molecules (version 4.0) are summarized in Table 1. CRR-related regions and CRR-unrelated regions within the genomic loci are depicted as red rectangles and thick black lines, respectively. The horizontal black arrows show the position and orientation of LTRs. The orientation of sequences lacking LTR is depicted by white arrows. The vertical black arrows mark the position of A-rich regions, which likely represent false poly(A) tails in corresponding fl-cDNA sequences. The imperfect tandem repeat present in the genomic locus "I" is depicted as an array of yellow arrowheads (note that their number does not reflect the number of monomers).

The fl-cDNA sequences were used to search the genome sequence of Oryza sativa cv. Nipponbare to find corresponding genomic loci. The identified genomic loci shared 99.7–100% sequence similarity with the fl-cDNAs. Thirteen genomic sequences were interrupted by one to nine introns, which were identified as regions missing in the fl-cDNA sequences and bordered by the canonical GT-AG dinucleotides. The intron spliced out from sequence AK102311 was extremely long (12,999 bp) and was composed largely of a cluster of an unknown tandem repeat (locus I in Figure 2). Comparison of multiple fl-cDNAs derived from the same genomic loci revealed alternative splicing and variability in the position of poly(A) tails (loci A, B, E, and O in Figure 2). The genomic loci were also analyzed with respect to the CRR sequences. Full-length elements were identified in eight loci. The remaining genomic loci contained solo LTR or truncated CRR sequences. The similarity between two LTRs of the full-length elements varied between 90.33% and 99.91%. Considering that 5'- and 3'-LTRs are identical upon insertion and that the rate of nucleotide substitution is 6.5 x 10^–9 (GAUT et al. 1996), the age of these CRR elements was estimated to be from 0.1 to 15 MY.

The search in the EST database in the GenBank revealed 55 accessions (of 406,790 ESTs) with sequence similarity to CRRs. Most of them (38) were similar to the noaCRR1 subfamily. Six sequences shared similarity with CRR1, six with CRR2, and five with noaCRR2 subfamilies. About half of the ESTs had a chimerical structure containing CRR-unrelated sequences. CRR-related regions mostly corresponded to the LTR or the 5'-UTR regions (supplemental Table 4 at http://www.genetics.org/supplemental/). The internal coding region was present in only eight ESTs and it was exclusively represented by a short sequence upstream of the 3'-LTR. Only six EST sequences (11%) originated from plants grown under normal physiological conditions, whereas most of them (78%) were from stressed or transgenic plants or from callus tissue (supplemental Table 4 at http://www.genetics.org/supplemental/). No EST sequence was identical to any of the KOME fl-cDNAs.

Experimental confirmation of CRR transcription:
RT–PCR was used to assess the transcriptional patterns of the CRR elements in different rice organs (root, leaf, and panicles) and to exploit transcripts associated with the internal regions of CRRs, which were not found in the fl-cDNA/EST databases. RT–PCR was performed using primers specific for three CRR subfamilies, including CRR1, CRR2, and noaCRR1 (supplemental Table 1 at http://www.genetics.org/supplemental/). These primers covered the entire internal region of individual CRR elements in 0.5- to 1-kb intervals. Since the similarity in the pol region is very high between CRR1 and CRR2, we used the same primer pairs to cover this region for both subfamilies.

RT–PCR products of expected sizes were obtained in all reactions and showed the same pattern in three different organs (Figure 3). However, there were differences in yield in many cases, mostly showing a higher abundance of CRR transcripts in panicles than in roots and leaves (Figure 3, A and B). As the actin control showed a similar yield in all three organs, those differences were not due to unequal RNA loading. One considerably shorter fragment was amplified in addition to the fragment of expected size using primers CRR1/2_2F and CRR1/2_3R (Figure 3A). Negative controls, which were included for each primer combination and each RNA sample, yielded no products. RT–PCR products amplified from leaf and in few cases from root and panicle were cloned and sequenced. All of the obtained sequences (64 total; supplemental Table 2 at http://www.genetics.org/supplemental/) shared high similarity with CRR elements. Most sequences derived from the coding region contained one intact open reading frame (ORF), which could be directly translated into protein. The putative protein sequences contained motifs of active sites, which suggests that they could be functional (data not shown).

View larger version (52K): [in this window] [in a new window] [Download PPT slide]   FIGURE 3.— RT–PCR analysis of CRR transcription. RT–PCR was performed using 14 primer pairs specific to both CRR1 and CRR2 subfamilies (A); to CRR1 only (B); to CRR2 only (C); and to noaCRR1 only (D). RNA isolated from roots (R), leaves (L), or panicles (P) was used as a template. RT–PCR with primers specific for the actin gene was carried out as an RNA loading control (E). The approximate locations of the primers within the CRR elements are marked in approximate positions of amplified regions within autonomous (CRR1 and CRR2) and nonautonomous (noaCRR1) elements (F). Only one scheme is shown for both autonomous CRR elements, CRR1 and CRR2, because their structure is the same. Primer combinations were as follows: 1—CRR1/2_1F and CRR1/2_2R; 2—CRR1/2_2F and CRR1/2_3R; 3—CRR1/2_3F and CRR1/2_4R; 4—CRR1/2_4F and CRR_ppt1R; 5—CRR1_2F and CRR1_3R; 6—CRR1_3F and CRR1_4R; 7—CRR1_4F and 5R; 8—CRR2_2F and CRR2_3R; 9—CRR2_3F and CRR2_4R; 10—CRR2_4F and CRR2_5R; 11—noaCRR1_2F and noaCRR1_3R; 12—noaCRR1_6F + noaCRR1_7Rb; 13—noaCRR1_4F + noaCRR1_5R; 14—noaCRR1_5F and CRR_ppt1R.

View larger version (79K): [in this window] [in a new window] [Download PPT slide]   FIGURE 4.— Detection of CRR transcription and CRR smRNAs by Northern blot hybridization. (A) Detection of CRR transcripts using five different probes derived from CRR1, CRR2, and noaCRR1. RNA ladder between 0.24 and 9.9 kb was used as a marker (M). Signals were detected only with CRR2 LTR and the two noaCRR1 probes, which hybridized to an 3.1-kb transcript (solid arrowheads) and as a smear between 4 and 15 kb. (B) Low-molecular-weight RNA was separated on 15% polyacrylamide gel. A faint band corresponding to the smRNA is marked by an open arrowhead. (C) Blots were hybridized with four CRR probes. The noaCRR1 probe hybridized strongly to the smRNA of 23–24 nt. The CRR1 probe generated several faint hybridization signals to the smRNA of 21–25 nt. The CRR2 LTR and P98-A8 probes did not hybridize with the smRNA. Probe OsmiR156 was used to check RNA quality on blots.

View larger version (91K): [in this window] [in a new window] [Download PPT slide]   FIGURE 5.— Mapping ends of CRR transcripts using RACE. (Left) 3'-RACE products. (Right) 5'-RACE products. DNA from bands marked by arrowheads was cloned.

In addition to the experimental detection of CRR-derived smRNA, we also searched for CRR-related smRNAs in rice databases (SUNKAR et al. 2005b; JOHNSON et al. 2007). The search within the databases of SUNKAR et al. (2005b) and JOHNSON et al. (2007) revealed 1 and 25 sequences, respectively, with high similarity to CRR elements (supplemental Table 5 at http://www.genetics.org/supplemental/). Of these smRNAs, 1 was derived from CRR1, 4 from CRR2, 17 from noaCRR1, and 4 from noaCRR2. The majority (88%) of smRNA sequences originated from the LTR and 5'-UTR regions (supplemental Table 5 at http://www.genetics.org/supplemental/). This is in agreement with the fact that these two regions were most represented among the fl-cDNA and EST sequences described above. Some smRNA sequences showed a perfect match to the fl-cDNA and EST sequences but it was not possible to determine whether they originated from these sequences because they have multiple identical matches to different genomic loci. The only exception was smRNA15884, which matches only one locus in the entire rice genome. All identified CRR-derived smRNAs occurred only one to two times among 35,454 in the genome-mapped smRNA collection (supplemental Table 5 at http://www.genetics.org/supplemental/ and JOHNSON et al. 2007). Importantly, five smRNA sequences shared significance with the noaCRR1 and 1 with the CRR1 probes that hybridized to the smRNA in Northern hybridization (Figure 4). In contrast, no smRNA sequence was similar to the CRR2 probe, which did not hybridize to the smRNA. Thus, our experimental data are in agreement with the result of the computational analysis of CRR-derived smRNA sequences.

Splicing of CRR sequences:
Two spliced regions were found in the CRR1 sequences derived from RT–PCR and 3'-RACE analyses. The first one was identified as a missing region in the shorter fragment amplified by primers CRR1/2_2F and CRR1/2_3R (Figure 3A). The missing region proved to be an intron because it was bordered by canonical 5'GT and AG 3' dinucleotides. This was also confirmed by absence of the spliced-size product when the genomic DNA was used as a template for PCR (data not shown). The splicing of this region resulted in a complete removal of the RT-coding domain predicted by the RPS-Blast program. The position of the acceptor site was the same in all analyzed sequences. However, there were three different donor sites (DS1–3) located very close to one another and alternatively used for the splicing (Figure 6). As the donor sites were placed in three different reading frames, the impact of the splicing on the translation of the downstream coding domain (RNaseH, integrase, chromodomain) differed. The splicing at the site DS2 removed only the RT-coding domain but still allowed the translation of the downstream domains, whereas the splicing at sites DS1 and DS3 separated upstream and downstream coding regions into different reading frames and most likely suppressed translation of the downstream domains. Although all spliced sequences had higher similarity to CRR1 than to the CRR2 subfamily, comparison of this region showed that donor and acceptor sites are conserved in all CRR subfamilies and also in the related CRM (maize) and Cereba (barley) elements (Figure 6).

View larger version (77K): [in this window] [in a new window] [Download PPT slide]   FIGURE 6.— Alignment of sequences of centromeric retrotransposons in the region spliced in CRR1 transcripts. DV468781–DV468790 are GenBank accession nos. of sequences derived from the RT–PCR products. Donor (GT; marked DS1–DS3) and acceptor (AG) splice sites detected in CRR1 and found in other CR sequences are shown against a solid background. Shading marks additional GT and AG dinucleotides predicted as potential splice sites using the FSPLICE program. The reverse-transcriptase domain identified within the putative protein sequence of CRR1-CH10 using RPS-Blast (MARCHLER-BAUER et al. 2003) is in boldface type and underlined. Asterisks mark the positions of fully conserved nucleotides. Dots indicate parts of the intron sequences that are not shown in the alignment.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   FIGURE 7.— LTR sequences are spliced in the chromodomain-coding region. (A) Alignment of DNA sequences. Donor (GT) and acceptor (AG) splice sites are shown against a solid background. Asterisks mark the positions of fully conserved nucleotides. Dots indicate parts of the intron sequences that are not shown in the alignment. (B) Alignment of chromodomain sequences from different families of centromeric retrotransposons. The chromodomain in the Cereba protein defined by KORDIS (2005) is boxed. Parts missing in putative proteins translated from spliced CRR1 and CRR2 sequences are in boldface type.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
Transcription of the CRR elements:
The rice genome is occupied by many families of LTR retrotransposons (GAO et al. 2004). However, the transcriptional or transpositional activities are largely unknown for most rice retrotransposons. Transcripts derived from the RIRE9 family were detected in leaves and stems but not in roots (LI et al. 2000). Retrotransposon-related sequences belonging to several other families were found in the rice EST database (VICIENT et al. 2001a; JIANG et al. 2002). Retrotransposons are generally silent, but can be activated by various stress conditions (WESSLER 1996; GRANDBASTIEN 1998). Few rice retrotransposon families (Tos10, Tos17–20) are transpositionally active under tissue culture conditions (HIROCHIKA et al. 1996; HIROCHIKA 1997). Nevertheless, it has been demonstrated in several species that some retrotransposon families, or rather only some of their copies, are transcriptionally active (SUONIEMI et al. 1997; MEYERS et al. 2001; ROSSI et al. 2001; VICIENT et al. 2001a,b; ECHENIQUE et al. 2002; NEUMANN et al. 2003, 2005), which suggests that they can escape from the host silencing mechanisms.

CRR transcripts were detected by RT–PCR in all three organs tested (root, leaf, panicle), suggesting that the CRR-related sequences are transcribed constitutively. Although transcription can be initiated from promoters located within 5'-LTRs (supplemental Figure 1 at http://www.genetics.org/supplemental/), analysis of the fl-cDNA sequences showed that most CRR transcripts are probably initiated from promoters upstream of the CRR elements (Figure 2). The level of CRR transcription appeared to be low. The frequencies of CRR transcripts in the fl-cDNA and EST databases are 0.089% and 0.013%, respectively. Similar EST frequencies were reported for both Ty3/gypsy and Ty1/copia retrotransposons present in other Graminae species (VICIENT et al. 2001a; ECHENIQUE et al. 2002), suggesting that the CRR family does not differ significantly in transcriptional level from other retrotransposon families. The low level of transcription of the CRR sequences, except for a few specific loci, was confirmed by Northern blot hybridization (Figure 4A). We also found that many CRR transcripts were possibly derived from relatively few genomic elements. Several fl-cDNAs were derived from the same genomic loci (Figure 2). A total of 24 RT–PCR and RACE sequences had the highest similarity to the CRR2 element associated with fl-cDNA AY828096 (locus N in Figure 2). Six RT–PCR sequences were most similar to three different regions of one noaCRR1 element (DQ458290). All six CRR2 3'-RACE sequences seemed to originate from the same genomic locus (locus E in Figure 2). These results suggest that only a few CRR elements escape silencing and contribute to the transcription.

We mapped the chromosomal locations of the genomic loci corresponding to the fl-cDNAs (Table 1). Two loci were mapped to regions of chromosomes 3 and 10 that contain the centromere-specific satellite CentO arrays. Chromosomes 3 and 10 contain <500 kb of the CentO repeats (CHENG et al. 2002; YAN et al. 2006). These two loci are likely located within the CENH3-binding domains that span 750 kb in chromosome 8 and 1800 kb in chromosome 3 (NAGAKI et al. 2004; YAN et al. 2006). Nine other loci were located within 5 Mbp from the CentO arrays. The remaining eight loci were more distant (up to 21 Mbp) from the centromeres. These results show that the CRR elements located in the centromeric regions are not more frequently transcribed than those in the pericentromeric regions.

Possible function of the CRR transcripts:
We analyzed the potential coding capacity of the CRR-related fl-cDNAs. The longest ORF was considered a potential coding sequence. The length of such coding sequences varied from 99 to 2046 nt (Figure 2). Comparison of the positions between the longest ORFs and CRR-related regions showed that they were overlapping in 13 (52%) fl-cDNAs, which in some cases was reflected by similarity to CRR putative polyprotein sequences (data not shown). The remaining fl-cDNAs contained a CRR-related region exclusively upstream (2 fl-cDNAs) or downstream (10 fl-cDNAs) of the longest ORF. Proteins translated from these fl-cDNAs were used as queries for searches made by RPS-Blast (MARCHLER-BAUER et al. 2003) and InterProScan (ZDOBNOV and APWEILER 2001), but no significant similarity to any proteins with known function was found.

The role of partial and chimerical CRR transcripts can only be speculated. Some of them may encode for proteins of unknown function, while others may represent noncoding RNAs with regulatory functions (MOREY and AVNER 2004; COSTA 2005). Several recent reports demonstrated the function of noncoding RNA on heterochromatin structure and centromere function. MAISON et al. (2002) were the first to demonstrate that the higher-order structure of the centromeric heterochromatin in mice depends on the presence of an RNA component. In maize, transcripts ranging in size from 40 to 900 nt derived from CRM elements and centromeric satellite repeat CentC are tightly bound to CENH3, suggesting a potential role of such noncoding RNA in the specification of centromeric chromatin (TOPP et al. 2004). Most recently, BOUZINBA-SEGARD (2006) showed that centromeric RNAs transcribed from the centromeric minor satellite repeat of mice are localized to centromeres. Forced accumulation of the 120-nt transcripts leads to defects in chromosome segregation and sister-chromatid cohesion (BOUZINBA-SEGARD et al. 2006). The authors proposed that the centromeric RNAs may play a role in regulation of heterochromatin assembly by tethering of kinetochore- and heterochromatin-associated proteins.

Our PCR-based experiment could not prove the hypothesis that CRR promoters may play a significant role in transcription of the downstream sequences, such as CentO repeats, which are highly intermingled with CRR elements in rice centromeres (CHENG et al. 2002). Cotranscripts of CRR and CentO repeats neither were found in the sequence databases nor were detected using RT–PCR (data not shown). In addition, we did not detect transcripts by RT–PCR using primers designed from downstream sequences of specific CRR elements in 12 loci within the centromere of chromosome 8 (data not shown). Thus, although some CRR insertions can provide active promoters, as we showed by 5'-RACE experiments, their impact on transcription of the downstream sequences may be limited. This is in agreement with the result of MAY et al. (2005) who detected cotranscripts of the Arabidopsis thaliana centromeric satellite repeat cen180 and retrotransposon Athila (106B) in the met1 mutant but not in the wild-type plant.

We propose that the long transcripts and smRNA derived from CRR elements may contribute to the formation of centromeric heterochromatin in rice. One possible explanation for the low level of CRR transcription is that most CRR transcripts may be turned over quickly by the RNAi pathway or integrated into centromeric chromatin. Several recent studies reported that a significant number of transcripts derived from centromeric repeats accumulated in mutants of components of the RNAi machinery (VOLPE et al. 2002; FUKAGAWA et al. 2004; MURCHISON et al. 2005). Our data showed that there are considerable differences in abundance and origin of the smRNAs derived from individual CRR subfamilies. While the noaCRR1 transcripts seem to be extensively processed into smRNA, CRR1- and CRR2-derived smRNAs are much less abundant (Figure 4C; supplemental Table 5 at http://www.genetics.org/supplemental/). Strikingly, all identified small RNA sequences derived from CRR1, noaCRR1, and noaCRR2 elements originated from the LTR and 5'-UTR regions. In contrast, all CRR2 smRNAs originated from the inside part, including 5'-UTR and gag-pol coding regions. As we showed that all CRR regions are transcribed (Figures 3 and 5), these findings suggest a differential extent of RNAi processing of the CRR transcripts. CRR1 and CRR2 elements are almost exclusively located in the centromeric regions. However, noaCRR1 elements are distributed more widely in the rice genome and were found in both centromeric and noncentromeric regions (NAGAKI et al. 2005). The observed differences may also be due to differences in copy numbers of individual CRR elements in the rice genome and to differences in abundance of the corresponding transcripts. The absence of CRR2 LTR-derived smRNA correlates with the high abundance of long transcripts hybridizing with the CRR2 LTR probe (Figure 4), which suggests that some CRR2 transcripts escape from the RNAi processing. These results suggest that the different CRR subfamilies, and perhaps also different parts of their sequences, play different roles in the RNAi-mediated pathway for formation and maintenance of centromeric heterochromatin.

Alternative splicing of CRR elements:
Although retroviruses are spliced during post-transcriptional processing (RABSON and GRAVES 1997), splicing of LTR retrotransposons is rare and has been reported in only a few retrotransposon families (BRIERLEY and FLAVELL 1990; VICIENT et al. 2001b; NEUMANN et al. 2003). Retrotransposon splicing was proposed to play roles in regulation of the GAG/GAG-PRO-POL or GAG-PRO/GAG-PRO-POL ratios (BRIERLEY and FLAVELL 1990; NEUMANN et al. 2003) and in expression of the envelope-like gene (VICIENT et al. 2001b). The splicing of CRR transcripts is unique in that at least two regions, both of which are different from the spliced regions in the three above-mentioned retrotransposons, are spliced from autonomous CRR elements. CRR elements seem to encode all protein domains within a single ORF (NAGAKI et al. 2005) and thus cannot regulate the GAG (GAG-PRO) to GAG-PRO-POL ratio by the two common translational recoding mechanisms on the basis of stop-codon readthrough or ribosomal frameshifting (SWANSTROM and WILLS 1997; VOGT 1997; GAO et al. 2003). However, as GAG is generally required in a higher molar amount than POL (SWANSTROM and WILLS 1997), CRRs must use a strategy that favors expression of GAG or GAG-PRO over GAG-PRO-POL polyproteins. Considering that the splicing of the reverse-transcriptase-coding region in CRRs results in separation of gag-pro and pol genes into different reading frames, it is likely that translation of the pol gene from spliced transcripts is suppressed while the translation of gag and pro genes remains unaffected. As only some CRR transcripts are spliced, the ratio between GAG-PRO and GAG-PRO-POL polyproteins might be controlled by the ratio between spliced and unspliced transcripts. Interestingly, the splice sites appear to be conserved among all autonomous CRR subfamilies (including the newly discovered CRR3 subfamily; Figures 6 and 7) as well as in the centromeric retrotransposons from maize (CRM) and barley (Cereba), supporting our hypothesis that CRR splicing may play a role in regulation of the expression of its encoding genes.

	ACKNOWLEDGEMENTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
We thank Dan Voytas for his valuable comments on the manuscript. This research was supported by Department of Energy grant FG02-01ER15266 to J.J.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 

ALTSCHUL, S. F., T. L. MADDEN, A. A. SCHAFFER, J. H. ZHANG, Z. ZHANG et al., 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389–3402.[Abstract/Free Full Text]

BANNISTER, A. J., P. ZEGERMAN, J. F. PARTRIDGE, E. A. MISKA, J. O. THOMAS et al., 2001 Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410: 120–124.[CrossRef][Medline]

BERNSTEIN, E., and C. D. ALLIS, 2005 RNA meets chromatin. Genes Dev. 19: 1635–1655.[Abstract/Free Full Text]

BOGUSKI, M. S., T. M. J. LOWE and C. M. TOLSTOSHEV, 1993 Dbest: database for expressed sequence tags. Nat. Genet. 4: 332–333.[CrossRef][Medline]

BOUZINBA-SEGARD, H., A. GUAIS and C. FRANCASTEL, 2006 Accumulation of small murine minor satellite transcripts leads to impaired centromeric architecture and function. Proc. Natl. Acad. Sci. USA 103: 8709–8714.[Abstract/Free Full Text]

BRIERLEY, C., and A. J. FLAVELL, 1990 The retrotransposon Copia controls the relative levels of its gene products posttranscriptionally by differential expression from its two major mRNAs. Nucleic Acids Res. 18: 2947–2951.[Abstract/Free Full Text]

BRODIE, R., R. L. ROPER and C. UPTON, 2004 JDotter: a Java interface to multiple dotplots generated by dotter. Bioinformatics 20: 279–281.[Abstract/Free Full Text]

CAM, H. P., T. SUGIYAMA, E. S. CHEN, X. CHEN, P. C. FITZGERALD et al., 2005 Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome. Nat. Genet. 37: 809–819.[CrossRef][Medline]

CHAN, S. W. L., I. R. HENDERSON and S. E. JACOBSEN, 2005 Gardening the genome: DNA methylation in Arabidopsis thaliana. Nat. Rev. Genet. 6: 351–360.[CrossRef][Medline]

CHENG, Z. K., F. G. DONG, T. LANGDON, O. Y. SHU, C. R. BUELL et al., 2002 Functional rice centromeres are marked by a satellite repeat and a centromere-specific retrotransposon. Plant Cell 14: 1691–1704.[Abstract/Free Full Text]

COSTA, F. F., 2005 Non-coding RNAs: new players in eukaryotic biology. Gene 357: 83–94.[CrossRef][Medline]

DESHPANDE, G., G. CALHOUN and P. SCHEDL, 2005 Drosophila argonaute-2 is required early in embryogenesis for the assembly of centric/centromeric heterochromatin, nuclear division, nuclear migration, and germ-cell formation. Genes Dev. 19: 1680–1685.[Abstract/Free Full Text]

DURAND-DUBIEF, M., and P. BASTIN, 2003 TbAGOI, an Argonaute protein required for RNA interference, is involved in mitosis and chromosome segragation in Trypanosoma brucei. BMC Biol. 1: 2.[CrossRef][Medline]

ECHENIQUE, V., B. STAMOVA, P. WOLTERS, G. LAZO, V. L. CAROLLO et al., 2002 Frequencies of Ty1-copia and Ty3-gypsy retroelements within the Triticeae EST databases. Theor. Appl. Genet. 104: 840–844.[CrossRef][Medline]

ELGIN, S. C. R., 1996 Heterochromatin and gene regulation in Drosophila. Curr. Opin. Genet. Dev. 6: 193–202.[CrossRef][Medline]

FISCHLE, W., Y. M. WANG, S. A. JACOBS, Y. C. KIM, C. D. ALLIS et al., 2003 Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 bv Polvcomb and HP1 chromodomains. Genes Dev. 17: 1870–1881.[Abstract/Free Full Text]

FUKAGAWA, T., M. NOGAMI, M. YOSHIKAWA, M. IKENO, T. OKAZAKI et al., 2004 Dicer is essential for formation of the heterochromatin structure in vertebrate cells. Nat. Cell Biol. 6: 784–791.[CrossRef][Medline]

GAO, L. H., E. M. MCCARTHY, E. W. GANKO and J. F. MCDONALD, 2004 Evolutionary history of Oryza sativa LTR retrotransposons: a preliminary survey of the rice genome sequences. BMC Genomics 5: 18.[CrossRef][Medline]

GAO, X., E. R. HAVECKER, P. V. BARANOV, J. F. ATKINS and D. F. VOYTAS, 2003 Translational recoding signals between gag and pol in diverse LTR retrotransposons. RNA 9: 1422–1430.[Abstract/Free Full Text]

GAUT, B. S., B. R. MORTON, B. C. MCCAIG and M. T. CLEGG, 1996 Substitution rate comparisons between grasses and palms: synonymous rate differences at the nuclear gene Adh parallel rate differences at the plastid gene rbcL. Proc. Natl. Acad. Sci. USA 93: 10274–10279.[Abstract/Free Full Text]

GENDREL, A. V., and V. COLOT, 2005 Arabidopsis epigenetics: when RNA meets chromatin. Curr. Opin. Plant Biol. 8: 142–147.[CrossRef][Medline]

GORINSEK, B., F. GUBENSEK and D. KORDIS, 2004 Evolutionary genomics of chromoviruses in eukaryotes. Mol. Biol. Evol. 21: 781–798.[Abstract/Free Full Text]

GORINSEK, B., F. GUBENSEK and D. KORDIS, 2005 Phylogenomic analysis of chromoviruses. Cytogenet. Genome Res. 110: 543–552.[CrossRef][Medline]

GRANDBASTIEN, M. A., 1998 Activation of plant retrotransposons under stress conditions. Trends Plant Sci. 3: 181–187.[CrossRef]

GREWAL, S. I. S., and J. C. RICE, 2004 Regulation of heterochromatin by histone methylation and small RNAs. Curr. Opin. Cell Biol. 16: 230–238.[CrossRef][Medline]

HIROCHIKA, H., 1997 Retrotransposons of rice: their regulation and use for genome analysis. Plant Mol. Biol. 35: 231–240.[CrossRef][Medline]

HIROCHIKA, H., K. SUGIMOTO, Y. OTSUKI, H. TSUGAWA and M. KANDA, 1996 Retrotransposons of rice involved in mutations induced by tissue culture. Proc. Natl. Acad. Sci. USA 93: 7783–7788.[Abstract/Free Full Text]

HUDAKOVA, S., W. MICHALEK, G. G. PRESTING, R. TEN HOOPEN, K. DOS SANTOS et al., 2001 Sequence organization of barley centromeres. Nucleic Acids Res. 29: 5029–5035.[Abstract/Free Full Text]

JENUWEIN, T., and C. D. ALLIS, 2001 Translating the histone code. Science 293: 1074–1080.[Abstract/Free Full Text]

JIANG, N., I. K. JORDAN and S. R. WESSLER, 2002 Dasheng and RIRE2: a nonautonomous long terminal repeat element and its putative autonomous partner in the rice genome. Plant Physiol. 130: 1697–1705.[Abstract/Free Full Text]

JOHNSON, C., L. BOWMAN, A. T. ADAI, V. VANCE and V. SUNDARESAN, 2007 CSRDB: a small RNA integrated database and browser resource for cereals. Nucleic Acids Res. 35: D829–D833.[CrossRef][Medline]

KANELLOPOULOU, C., S. A. MULJO, A. L. KUNG, S. GANESAN, R. DRAPKIN et al., 2005 Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev. 19: 489–501.[Abstract/Free Full Text]

KELLOGG, E. A., 2001 Evolutionary history of the grasses. Plant Physiol. 125: 1198–1205.[Free Full Text]

KIKUCHI, S., K. SATOH, T. NAGATA, N. KAWAGASHIRA, K. DOI et al., 2003 Collection, mapping, and annotation of over 28,000 cDNA clones from japonica rice. Science 301: 376–379.[Abstract/Free Full Text]

KORDIS, D., 2005 A genomic perspective on the chromodomain-containing retrotransposons: chromoviruses. Gene 347: 161–173.[CrossRef][Medline]

LEE, H. R., P. NEUMANN, J. MACAS and J. JIANG, 2006 Transcription and evolutionary dynamics of the centromeric satellite repeat CentO in rice. Mol. Biol. Evol. 23: 2505–2520.[Abstract/Free Full Text]

LI, Z. Y., S. Y. CHEN, X. W. ZHENG and L. H. ZHU, 2000 Identification and chromosomal localization of a transcriptionally active retrotransposon of Ty3-gypsy type in rice. Genome 43: 404–408.[Medline]

MAISON, C., D. BAILLY, A. H. F. M. PETERS, J. P. QUIVY, D. ROCHE et al., 2002 Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component. Nat. Genet. 30: 329–334.[CrossRef][Medline]

MARCHLER-BAUER, A., J. B. ANDERSON, C. DEWEESE-SCOTT, N. D. FEDOROVA, L. Y. GEER et al., 2003 CDD: a curated Entrez database of conserved domain alignments. Nucleic Acids Res. 31: 383–387.[Abstract/Free Full Text]

MATZKE, M. A., and J. A. BIRCHLER, 2005 RNAi-mediated pathways in the nucleus. Nat. Rev. Genet. 6: 24–35.[CrossRef][Medline]

MAY, B. P., Z. B. LIPPMAN, Y. D. FANG, D. L. SPECTOR and R. A. MARTIENSSEN, 2005 Differential regulation of strand-specific transcripts from Arabidopsis centromeric satellite repeats. PLoS Genet. 1: 705–714.

MEYERS, B. C., S. V. TINGLEY and M. MORGANTE, 2001 Abundance, distribution, and transcriptional activity of repetitive elements in the maize genome. Genome Res. 11: 1660–1676.[Abstract/Free Full Text]

MILLER, J. T., F. G. DONG, S. A. JACKSON, J. SONG and J. M. JIANG, 1998 Retrotransposon-related DNA sequences in the centromeres of grass chromosomes. Genetics 150: 1615–1623.[Abstract/Free Full Text]

MOREY, C., and P. AVNER, 2004 Employment opportunities for non-coding RNAs. FEBS Lett. 567: 27–34.[CrossRef][Medline]

MURCHISON, E. P., J. F. PARTRIDGE, O. H. TAM, S. CHELOUFI and G. J. HANNON, 2005 Characterization of Dicer-deficient murine embryonic stem cells. Proc. Natl. Acad. Sci. USA 102: 12135–12140.[Abstract/Free Full Text]

NAGAKI, K., and M. MURATA, 2005 Characterization of CENH3 and centromere-associated DNA sequences in sugarcane. Chromosome Res. 13: 195–203.[CrossRef][Medline]

NAGAKI, K., J. Q. SONG, R. M. STUPAR, A. S. PAROKONNY, Q. P. YUAN et al., 2003 Molecular and cytological analyses of large tracks of centromeric DNA reveal the structure and evolutionary dynamics of maize centromeres. Genetics 163: 759–770.[Abstract/Free Full Text]

NAGAKI, K., Z. K. CHENG, S. OUYANG, P. B. TALBERT, M. KIM et al., 2004 Sequencing of a rice centromere uncovers active genes. Nat. Genet. 36: 138–145.[CrossRef][Medline]

NAGAKI, K., P. NEUMANN, D. F. ZHANG, S. OUYANG, C. R. BUELL et al., 2005 Structure, divergence, and distribution of the CRR centromeric retrotransposon family in rice. Mol. Biol. Evol. 22: 845–855.[Abstract/Free Full Text]

NEUMANN, P., D. POZARKOVA and J. MACAS, 2003 Highly abundant pea LTR retrotransposon Ogre is constitutively transcribed and partially spliced. Plant Mol. Biol. 53: 399–410.[CrossRef][Medline]

NEUMANN, P., D. POZARKOVA, A. KOBLIZKOVA and J. MACAS, 2005 PIGY, a new plant envelope-class LTR retrotransposon. Mol. Genet. Genomics 273: 43–53.[CrossRef][Medline]

PAL-BHADRA, M., B. A. LEIBOVITCH, S. G. GANDHI, M. RAO, U. BHADRA et al., 2004 Heterochromatin silencing and HP1 localization in Drosophila are dependent on the RNAi machinery. Science 303: 669–672.[Abstract/Free Full Text]

PIDOUX, A. L., and R. C. ALLSHIRE, 2005 The role of heterochromatin in centromere function. Philos. Trans. R. Soc. Lond. B Biol. Sci. 360: 569–579.[CrossRef][Medline]

PRESTING, G. G., L. MALYSHEVA, J. FUCHS and I. SCHUBERT, 1998 A TY3/GYPSY retrotransposon-like sequence localizes to the centromeric regions of cereal chromosomes. Plant J. 16: 721–728.[CrossRef][Medline]

RABSON, A. B., and B. J. GRAVES, 1997 Synthesis and processing of viral RNA, pp. 205–261 in Retroviruses, edited by J. M. COFFIN, S. H. HUGHES and H. E. VARMUS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

RICE, P., I. LONGDEN and A. BLEASBY, 2000 EMBOSS: The European molecular biology open software suite. Trends Genet. 16: 276–277.[CrossRef][Medline]

ROSSI, M., P. G. ARAUJO and M. A. VAN SLUYS, 2001 Survey of transposable elements in sugarcane expressed sequence tags (ESTs). Genet. Mol. Biol. 24: 147–154.

SONNHAMMER, E. L. L., and R. DURBIN, 1995 A dot-matrix program with dynamic threshold control suited for genomic DNA and protein sequence analysis. Gene 167: 1–10.[CrossRef][Medline]

STADEN, R., 1996 The Staden sequence analysis package. Mol. Biotechnol. 5: 233–241.[Medline]

SUNKAR, R., T. GIRKE, P. K. JAIN and J. K. ZHU, 2005a Cloning and characterization of MicroRNAs from rice. Plant Cell 17: 1397–1411.[Abstract/Free Full Text]

SUNKAR, R., T. GIRKE and J. K. ZHU, 2005b Identification and characterization of endogenous small interfering RNAs from rice. Nucleic Acids Res. 33: 4443–4454.[Abstract/Free Full Text]

SUONIEMI, A., D. SCHMIDT and A. H. SCHULMAN, 1997 BARE-1 insertion site preferences and evolutionary conservation of RNA and cDNA processing sites. Genetica 100: 219–230.[CrossRef][Medline]

SWANSTROM, R., and J. W. WIL