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.

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THE centromeric retrotransposon of rice (CRR) is highly enriched in the centromeres of rice chromosomes (CHENG et al. 2002; NAGAKI et al. 2005). CRR is a Ty3/gypsy-like retrotransposon (Metaviridae) and belongs to a class of chromoviruses (GORINSEK et al. 2004, 2005; KORDIS 2005). This class of retrotransposon is defined by the presence of a chromodomain within its integrase and seems to be widespread among eukaryotes. The chromodomain identified within chromoviruses is related to the chromodomain of heterochromatin protein 1 (HP1) of Drosophila melanogaster (GORINSEK et al. 2005; KORDIS 2005), which interacts with histone 3 methylated at lysine 9 (H3K9me) (BANNISTER et al. 2001; FISCHLE et al. 2003). Both HP1 and H3K9me are hallmarks of heterochromatin (ELGIN 1996; GREWAL and RICE 2004). Although it has not been experimentally proved yet, it is likely that the chromodomain of the CRR elements is responsible for their preferential localization in the centromeres as well as in pericentromeric regions.

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.

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.

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.

 

Bioinformatic analysis of transcribed CRR sequences in databases:

To find if CRR elements are transcribed in the rice genome, we first searched the KOME (fl-cDNA) (KIKUCHI et al. 2003) and GenBank EST (BOGUSKI et al. 1993) databases. Sequences typical for each of four CRR subfamilies (CRR1, CRR2, noaCRR1, and noaCRR2) described by NAGAKI et al. (2005) were used as queries. We found 25 CRR-like fl-cDNA sequences (from a total of 28,469), which originated from natural plant tissues or callus (supplemental Table 3 at http://www.genetics.org/supplemental/). Of these fl-cDNA sequences, 7 belonged to CRR2, 14 to noaCRR1, and only 1 to noaCRR2 subfamilies (Figure 2; supplemental Table 3 at http://www.genetics.org/supplemental/). The remaining 3 fl-cDNA sequences had chimerical structures containing sequences derived from different CRR subfamilies or from CRR3, a previously uncharacterized subfamily.

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).

 

View this table: In this window In a new window   TABLE 1 Distances of the transcribed CRR loci from centromeres

 
Only two fl-cDNA sequences, AK064474 and AK106612, appeared to contain exclusively CRR-related sequences. These two fl-cDNAs had a similar structure, containing the 3'-part of the coding region, PPT, and an incomplete 3'-LTR (loci J and Q in Figure 2). All other fl-cDNA accessions (23 of 25; 92%) were chimerical transcripts containing both CRR-related and CRR-unrelated sequences. Most of the CRR-related regions corresponded to the LTRs which, in some cases, were extended into the 5'-UTR or preceded by the coding region (Figure 2). None of the fl-cDNAs contained the complete polyprotein-coding region. The CRR-unrelated regions were always located upstream of the CRR-related regions, indicating that upstream, not CRR-related promoters, drove the transcription of these sequences. Seventeen fl-cDNA sequences terminated in the CRR-related regions and 8 extended into downstream CRR-unrelated regions. Six fl-cDNA sequences terminated within the A-rich region in the 5'-UTR of CRR sequences, which likely worked as a false poly(A) tail during reverse transcription. Interestingly, the majority of fl-cDNA sequences (22 of 25; 88%) was derived from the plus strand of the CRR elements.

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.

 
To examine the abundance and size variability of various CRR transcripts described above, we carried out Northern blot hybridizations using RNA isolated from leaves. Blots containing 40 µg of total RNA were hybridized with probes specific to the LTR region of CRR1, CRR2, and noaCRR1, the RT-coding region of CRR1 and CRR2, and the GAG-coding region of noaCRR1. Although all probes were prepared from RT–PCR or RACE clones (see below), strong and unambiguous signals were detected only by the CRR2 LTR probe and the two noaCRR1 probes (Figure 4A). These three probes hybridized to an 3.1-kb long transcript. The CRR2 LTR probe hybridized to this transcript strongly. However, both noaCRR1 probes hybridized weakly (Figure 4A). We assume that the transcript likely originated from a CRR2 element and that the weak hybridization to the noaCRR1 probes was due to partial similarity between CRR2 and noaCRR1 sequences. Its size, which is considerably shorter than would be expected for a hypothetical full-length CRR2 transcript (7–8 kb), suggests that the transcript was prematurely terminated, spliced, or originated from a truncated element. In addition to this transcript, the three probes also hybridized as a 4- to 15-kb smear having the highest intensity between 7 and 10 kb. The Northern hybridization results revealed a low level of transcription, suggesting that most of the CRR elements are transcriptionally silent.

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.

 

Mapping of 5'- and 3'-ends of CRR transcripts:

Although database searches and RT–PCR results showed that CRR sequences are transcribed in the rice genome, it remained unclear whether CRR transcription can be initiated from the CRR promoters and whether there are termination (polyadenylation) sites additional to those identified in some fl-cDNA and EST sequences. To address these questions, we conducted 5'- and 3'-RACE analyses using RNA isolated from leaves. Both RACE methods resulted in amplification of one or more major products for each of three CRR subfamilies (Figure 5). These products were cloned and several randomly selected clones were sequenced. The analysis of 17 3'-RACE and 27 5'-RACE sequences revealed several sites of transcription initiation as well as termination within each CRR subfamily (supplemental Figure 1 at http://www.genetics.org/supplemental/). Transcription initiations were found within the LTR but also in sequences located upstream of CRR elements. On the other hand, all polyadenylation sites were found within the LTR sequences. Importantly, some 5'-RACE and 3'-RACE products contained overlapping sequences. Since the overlap sequences between 5'- and 3'-LTRs in retrotransposon transcripts are crucial for replication (WILHELM and WILHELM 2001), this finding suggests that some CRR transcripts are capable of replication.

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.

 

CRR transcripts are processed into smRNA:

To find if CRR transcripts are processed into smRNA, we conducted Northern hybridizations using blots containing low-molecular-weight RNA isolated from rice leaves (Figure 4B). The blots were hybridized with the CRR-specific probes derived from LTRs (see MATERIALS AND METHODS). The noaCRR1 probe hybridized strongly to the smRNA of 23–24 nt in length (Figure 4C). Faint smRNA signals were also detected with the CRR1 probe. However, the CRR2 probes did not hybridize with smRNAs. The CRR2 LTR probe detected an 3.1-kb CRR transcript on the total RNA blots (Figure 4A), which implies that the majority, if not all, of CRR2 transcripts escaped processing by the RNAi machinery in rice leaves. To check RNA quality, the blots were reprobed with a probe specific for previously described miRNA OsmiR156 (SUNKAR et al. 2005a). Both sizes of this miRNA (20 and 21 nt) were consistently detected, showing that the small RNA on all blots was intact (Figure 4C).

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.

 
The second spliced region was found in the chromodomain-coding region in the 3'-LTR of CRR1 elements. It was detected in one 3'-RACE (DV468825) and two EST (CA998740 and CA999991) sequences as a deletion between donor and acceptor splice sites predicted by FSPLICE program (Figure 7). Splice-site analysis of the corresponding region in CRR2 sequences revealed the same donor but a different acceptor site. One fl-cDNA CRR2 sequence (AK121472) was spliced at the same donor site, which suggests that CRR2 transcripts were spliced in the same region as CRR1. The GT dinucleotide at the donor site was fully conserved among other sequences of centromeric retrotransposons (Figure 7). However, the AG dinucleotide in both of the two acceptor sites was present in CRR and CRM but absent in Cereba sequences.

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.

 
Comparison of the fl-cDNA AK067185 with the corresponding genomic sequence revealed a splicing event in the coding region of noaCRR1 (locus F in Figure 2; supplemental Figure 2 at http://www.genetics.org/supplemental/). This event removed the entire GAG-coding domain, but allowed potential translation of the downstream coding sequence (data not shown). Although we identified only one sequence spliced in this region, donor and acceptor sites were conserved in 16 of 26 noaCRR1 elements analyzed (supplemental Figure 2 at http://www.genetics.org/supplemental/), which suggests that their transcripts may also undergo splicing. Two additional fl-cDNAs (AK064150 and AK111287) were spliced within the 5'-LTR at the same acceptor site, which seems to be highly conserved among noaCRR1 elements (data not shown). However, as the donor sites were located at different positions in CRR-unrelated sequences, it is not clear whether full-size noaCRR1 transcripts can be spliced in the LTR as well.

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.

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.

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Communicating editor: V. SUNDARESAN

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