Present address: Institut de Génétique Moléculaire de Montpellier, CNRS-UMR 5535, 1919 Route de Mende, 34293 Montpellier-Cedex 5, France.
AtNUFIP, an essential protein for plant development, reveals the impact of snoRNA gene organisation on the assembly of snoRNPs and rRNA methylation in Arabidopsis thaliana
Article first published online: 24 JAN 2011
© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd
The Plant Journal
Volume 65, Issue 5, pages 807–819, March 2011
How to Cite
Rodor, J., Jobet, E., Bizarro, J., Vignols, F., Carles, C., Suzuki, T., Nakamura, K. and Echeverría, M. (2011), AtNUFIP, an essential protein for plant development, reveals the impact of snoRNA gene organisation on the assembly of snoRNPs and rRNA methylation in Arabidopsis thaliana. The Plant Journal, 65: 807–819. doi: 10.1111/j.1365-313X.2010.04468.x
- Issue published online: 24 FEB 2011
- Article first published online: 24 JAN 2011
- Accepted manuscript online: 24 DEC 2010 10:09AM EST
- Received 3 September 2010; revised 6 December 2010; accepted 18 December 2010; published online 24 January 2011.
- snoRNP assembly;
- atnuf mutants;
- rRNA methylation
- Top of page
- Experimental procedures
- Supporting Information
In all eukaryotes, C/D small nucleolar ribonucleoproteins (C/D snoRNPs) are essential for methylation and processing of ribosomal RNAs. They consist of a box C/D small nucleolar RNA (C/D snoRNA) associated with four highly conserved nucleolar proteins. Recent data in HeLa cells and yeast have revealed that assembly of these snoRNPs is directed by NUFIP protein and other auxiliary factors. Nevertheless, the precise function and biological importance of NUFIP and the other assembly factors remains unknown. In plants, few studies have focused on RNA methylation and snoRNP biogenesis. Here, we identify and characterise the AtNUFIP gene that directs assembly of C/D snoRNP. To elucidate the function of AtNUFIP in planta, we characterized atnufip mutants. These mutants are viable but have severe developmental phenotypes. Northern blot analysis of snoRNA accumulation in atnufip mutants revealed a specific degradation of C/D snoRNAs and this situation is correlated with a reduction in rRNA methylation. Remarkably, the impact of AtNUFIP depends on the structure of snoRNA genes: it is essential for the accumulation of those C/D snoRNAs encoded by polycistronic genes, but not by monocistronic or tsnoRNA genes. We propose that AtNUFIP controls the kinetics of C/D snoRNP assembly on nascent precursors to overcome snoRNA degradation of aberrant RNPs. Finally, we show that AtNUFIP has broader RNP targets, controlling the accumulation of scaRNAs that direct methylation of spliceosomal snRNA in Cajal bodies.
- Top of page
- Experimental procedures
- Supporting Information
Small nucleolar RNAs (snoRNAs) represent an abundant class of guide RNAs that direct modifications of approximately 200 residues on rRNAs in eukaryotes. Most snoRNAs fall into two families: (i) the box C/D snoRNAs, with conserved C and D motifs, which direct 2′-O-ribose methylations; and (ii) the box H/ACA snoRNAs, with conserved H/ACA motifs, which direct pseudouridylation. In addition, a subset of C/D and H/ACA snoRNAs is implicated in specific cleavage of rRNA precursors (pre-rRNA) (Bachellerie et al., 2002; Matera et al., 2007).
In vivo, each snoRNA associates with a set of core proteins, forming hundreds of nucleolar ribonucleoproteins (snoRNPs). The C/D snoRNAs associate with fibrillarin (Nop1p in yeast), which is the RNA methylase, NOP58, NOP56 and 15.5K (Snu13p in yeast). The H/ACA snoRNAs associate with dyskerin/NAP57 (Cbf5p in yeast), which is the pseudouridine synthase, NHP2, GAR1 and NOP10 (Brown et al., 2003a; Matera et al., 2007).
Another related class of guide RNAs are the small Cajal bodies RNAs (scaRNAs) that direct modifications of spliceosomal snRNAs in Cajal bodies (Darzacq et al., 2002). The scaRNAs contain C/D or H/ACA conserved elements but can also have hybrid C/D and H/ACA structure (Jády and Kiss, 2001). In vivo, scaRNAs associate with the same set of proteins as snoRNAs and form the corresponding scaRNPs.
In contrast with the conservation of snoRNP components, the plant snoRNA genes exhibit specific modes of genomic organization and expression (Brown et al., 2003a; Rodor et al., 2010). In animals, most snoRNAs are encoded within introns of protein coding genes and are produced by processing of the intron lariat. In plants, most snoRNAs are encoded by polycistronic clusters independently transcribed from their own promoter. They produce a polycistronic precursor that is processed by endonucleolytic and exonucleolytic trimming to release the individual snoRNAs. In plants there are also intronic snoRNAs, but most of them are in clusters. Plants also have a unique family of tRNA-snoRNA genes. These are transcribed by Pol III from the tRNA promoter producing a dicistronic precursor that is processed into a tRNA and a C/D snoRNA (Kruszka et al., 2003; Barbezier et al., 2009). Independent monocistronic snoRNA genes which are the majority in yeast are very rare in plants. The only clear examples are U3, which is transcribed by Pol III (Kiss et al., 1991), and U13, transcribed by Pol II (Kim et al., 2010). Notably, both U3 and U13 are implicated in pre-rRNA processing in mammals (Maxwell and Fournier, 1995; Cavailléet al., 1996).
All snoRNAs are produced by processing of a precursor (pre-snoRNA). Several studies in animals, yeast and plants have revealed a tight link between transcription, processing and assembly of the snoRNP on the nascent pre-snoRNA (Darzacq et al., 2006; Hirose et al., 2006; Barbezier et al., 2009). The initial processing steps are distinct on the monocistronic, intronic or polycistronic pre-snoRNAs. Nevertheless, the final steps of processing are similar and implicate 5′ and 3′ exonucleolytic trimming of flanking sequences to produce the mature snoRNA ends (Allmang et al., 1999; Van Hoof et al., 2000; Lee et al., 2003). Assembly of the snoRNP is essential to control exonucleolytic degradation and stabilize the mature snoRNA.
Assembly of C/D snoRNPs is initiated by the binding of 15.5K to the k-turn RNA motif formed by C/D elements (Watkins et al., 2000). Binding of 15.5K to the snoRNAs nucleates the recruitment of the three other core proteins forming the C/D snoRNP. Recent studies in yeast and human cells have revealed auxiliary factors that transiently interact with the core proteins and direct assembly of the snoRNP (McKeegan et al., 2007, 2009; Boulon et al., 2008).
A central factor directing assembly of C/D snoRNPs is NUFIP identified in HeLa cells. NUFIP directly interacts with the 15.5K protein, which binds to C/D snoRNA and facilitates the recruitment of the three other core proteins (McKeegan et al., 2007; Boulon et al., 2008).
In plants, snoRNP assembly factors have not been studied and their impact on snoRNA metabolism and development is not known. To address these questions, here we focus on the identification and characterization of the AtNUFIP gene in Arabidopsis thaliana and characterize two distinct atnufip mutant lines. Analysis of mutants shows that AtNUFIP is required for plant development and is essential for C/D snoRNA stability. Remarkably this effect depends on the genomic organization of snoRNAs and AtNUFIP is only essential for the accumulation of snoRNAs derived from polycistronic precursors. Finally, we show that AtNUFIP has an additional function and controls the biogenesis of C/D scaRNPs predicted to direct methylation of spliceosomal snRNAs.
- Top of page
- Experimental procedures
- Supporting Information
Identification of potential NUFIP homologs in plants
The human NUFIP and its homolog in yeast Rsa1p are divergent but share a short conserved motif PEP that directs the interaction with the 15.5K (Snu13p in yeast) core C/D snoRNP protein (Boulon et al., 2008). A TBLASTN search (Altschul et al., 1997) with the human PEP motif on the Arabidopsis thaliana and rice genomes identified a single gene encoding a protein with a PEP motif in each species (Figure 1a). Using the discovery motif MEME/MAST (Bailey and Elkan, 1994), we identified a second conserved motif in the C terminal extremity of plant putative NUFIP, human NUFIP and yeast Rsa1p (Figure 1a). This motif contains the CRM1-dependent nuclear export signal (NES) described in human NUFIP (Bardoni et al., 2003).
To further characterize the eukaryotic NUFIP family, we extended the previous analysis to several species, including protist and green unicellular algae (Figure 1b). In addition to PEP, the NES motif is conserved in most species, except in a few lower eukaryotes (Figure 1b). Alignment of this motif shows high conservation of hydrophobic residues that are important for nuclear export (Dong et al., 2009). In addition, the plant NUFIP proteins contain a third motif of unknown function, called motif P (Figure 1a,c), which is not found in any other protein. Finally, the zinc finger domain of NUFIP, which is related to a transcriptional function of the protein (Cabart et al., 2004), is specific to animal NUFIP.
This analysis shows that the central PEP motif associated with the C-terminal NES motif define the NUFIP family. In Arabidopsis thaliana, the predicted NUFIP homolog is encoded by gene At5g18440, hereafter called AtNUFIP.
Expression of AtNUFIP in Arabidopsis thaliana
The coding sequence of AtNUFIP gene was based on a partial cDNA. To eliminate any ambiguity, we mapped the 5′ and 3′ ends of AtNUFIP mRNA by RLM RACE. This mapping revealed two types of transcripts that differed by a 96 nucleotide intron retained on the 5′UTR (Figure 2a). Otherwise both transcripts have identical ORFs and 3′UTRs. Semi-quantitative RT-PCR on total RNA from wild type seedlings with primers flanking the retained intron (Figure 2a) confirmed that both transcripts are expressed at similar levels (Figure 2b).
We checked the expression of the AtNUFIP gene in different plant tissues by semi-quantitative RT-PCR. Primers were designed on the ORF extremities to amplify the two types of AtNUFIP mRNAs. These primers encompass several introns to distinguish PCR amplification from genomic DNA (Figure 2a). As a control we assessed expression of eEF1-α constitutive gene. AtNUFIP mRNA was specifically detected on all tissues (Figure 2c). Thus AtNUFIP is expressed constitutively but at a low level because a high number of PCR cycles was required to detect it as compared with eEF1-α mRNA.
AtNUFIP interacts directly with At15.5K
The PEP motif of AtNUFIP should direct its interaction with At15.5K. In Arabidopsis, At15.5K is encoded by three genes producing nearly identical proteins conserved with their human and yeast orthologues (Figure S1A). Considering that all three At15.5K genes are constitutively expressed (Figure S1B), we randomly chose At15.5K-3 (At4g22380) to test its interaction with AtNUFIP.
We first tested their interaction by yeast two-hybrid analysis. The AtNUFIP and At15.5K ORFs were cloned by fusion with the binding (BD) and activating (AD) domains of GAL4. As a negative control, we used the GAL4 AD or BD empty vector. Cells co-transformed with different combinations of these constructs were grown in the presence or absence of histidine selectable marker. Clearly, yeast cells co-transformed with AD-AtNUFIP and BD-At15.5K (or BD-AtNUFIP and AD-At15.5K) could grow in the absence of histidine, revealing an interaction between these two proteins (Figure 3a).
To confirm a direct interaction between AtNUFIP and At15.5K, we fixed a glutathione S-transferase (GST)-At15.5K recombinant protein to glutathione-sepharose beads to trap in vitro translated [35S]-Met-AtNUFIP. Bound and unbound products were analyzed by SDS-PAGE and revealed by autoradiography. [35S]-Met-AtNUFIP is highly susceptible to proteolysis and gives three major bands of approximately 50 kDa (Figure 3b). These bands were significantly enriched in the GST-At15.5K bound fraction and were not retained on control glutathione-GST beads (Figure 3b).
These results confirm that AtNUFIP directly interacts with At15.5K and could be implicated in C/D snoRNP assembly as human NUFIP.
AtNUFIP partially colocalizes with At15.5K at the nucleolar periphery
To determine the subcellular localization of AtNUFIP and At15.5K in vivo, we produced transgenic plants co-expressing GFP–AtNUFIP and At15.5K-RFP under the control of the constitutive 35S CaMV promoter. Several transgenic lines were produced expressing At15.5K-RFP in all tissues. The fluorescence was concentrated in the nucleolus, with major labeling in the center of this compartment, and in small foci associated to the nucleolus (Figure 4a). These foci correspond to Cajal bodies revealed by co-localization with U2B′′-GFP (Figure 4a), a specific marker of this compartment in plants (Collier et al., 2006).
Very few lines expressing GFP–AtNUFIP could be obtained. Moreover, in these lines, the GFP fluorescence was weak and restricted to root cells. GFP–AtNUFIP was localized in the nucleus with a diffused fluorescence in the nucleoplasm and a high concentration in the perinucleolar region (Figure 4b). In meristematic cells from root tips, the fluorescence was concentrated in the external layer of the nucleolus. In elongating root cells, the fluorescence concentrated in perinucleolar foci (Figure 4b). GFP–AtNUFIP was observed neither in the central region of the nucleolus nor in Cajal bodies (Figure 4b). The yellow signal in merged images shows that GFP–AtNUFIP partially co-localized with At15.5K-RFP in the external nucleolar layer and the perinucleolar foci (Figure 4b).
These results show partial co-localization of AtNUFIP with At15.5K in the nucleolar periphery and are in agreement with an interaction between AtNUFIP and At15.5K in vivo.
Characterization of T-DNA insertional mutant lines in the AtNUFIP gene
To study the function of AtNUFIP in vivo, we identified two T-DNA insertional mutant lines, the SALK 134962 and the GABI 680F01 lines, hereafter called atnuf-1 and atnuf-2 respectively. PCR genotyping confirmed that the insertions in atnuf-1 and atnuf-2 mutants are in the last exon and sixth intron respectively (Figure 2a). We obtained homozygous plants for atnuf-1 and atnuf-2 in the F2 generation by self crossing F1 heterozygous lines.
The atnuf-1 homozygous plants presented severe phenotypes but were fertile and could be maintained as homozygous line. A segregation test to confirm a single T-DNA insertion in the atnuf-1 mutant was not possible due to loss of the kanamycin resistance gene due to rearrangement of the T-DNA insertion. Complementation of atnuf-1 mutants with an AtNUFIP-FLAG-HA tagged gene fully rescued all phenotypes (Figure S2).
In atnuf-2 mutants, the GABI T-DNA insertion retained the sulfadiazine resistance gene, allowing confirmation of a single T-DNA insertion by a segregation test (result not shown). This was confirmed by full complementation of atnuf-2 mutants with the AtNUFIP-FLAG-HA tagged gene (Figure S2). The atnuf-2 mutants are sterile and are maintained in heterozygous state.
Full-length AtNUFIP mRNA detected by RT-PCR was not produced in both atnuf-1 and atnuf-2 lines (Figure 2b). Nevertheless, using primers upstream from the insertions (Figure 2a) ‘truncated’ mRNAs were detected in both mutants (Figure 2b). In both mutants the couple of primers 5F/R2 detected two mRNA forms distinguished by retention of the first intron (Figure 2b). Interestingly, in atnuf-2 a third major transcript was detected which corresponds to an unspliced product retaining both the first and second introns (Figure 2b). This indicates that splicing of AtNUFIP truncated transcript is affected in atnuf-2.
These data show that only truncated RNAs are expressed from atnuf-1 and atnuf-2 locus. If translated these would give truncated proteins (Figure 2a).
atnuf-1 and atnuf-2 have severe developmental phenotypes
Homozygous atnuf-1 and atnuf-2 mutants were affected in their development. Overall, both mutants had similar phenotypes but defects were enhanced in atnuf-2. Both mutants showed significant growth delay as compared with wild type plants (Figure 5a). Also, many atnuf-2 seedlings had premature growth arrest and did not reach the adult state. The atnuf-1 and atnuf-2 seedlings and adult plants were smaller and presented leaf morphological defects compared to wild type (Figure 5a). This situation was most evident in young seedlings that had a pointed leaf phenotype that is typical of ribosomal protein mutants (Van Lijsebettens et al., 1994). Both mutants had phyllotaxy defects revealed, for example, by some seedlings with three cotyledons or plants with two cauline leaves on the same node (Figure 5b). The atnuf-1 mutants had important floral defects and, in the case of atnuf-2, only few plants could develop flowers and in a limited number. In both mutants, flowers had lost organ symmetry, had altered number and morphology of petals and had shorter stamens (Figure 5c). Histological analysis of atnuf-1 mutants revealed a size reduction of the inflorescence meristem that presents a domed profile and leads to a premature termination (Figure 5d). Analysis of atnuf-1 pistil section showed a total disorganization and a reduced number of embryos (Figure 5e). Consequently fertility in atnuf-1 was highly reduced, with smaller siliques and few seeds, while atnuf-2 produced no seeds and was sterile (Figure 5f).
To unambiguously confirm that the phenotypes of atnuf-1 and atnuf-2 were due to the AtNUFIP gene insertion, we complemented the mutant lines with a tagged AtNUFIP–HA-FLAG gene containing all introns and driven from its own promoter. The complemented mutant plants showed complete rescue of the phenotypes (Figure S2).
Accumulation of C/D snoRNAs from polycistronic genes are reduced in atnuf mutants
Accumulation of snoRNAs in atnuf-1 and atnuf-2 mutants was analysed by Northern blots using radiolabelled oligonucleotide probes and total RNAs from wild type and atnuf seedlings (Figure 6). As a loading control, we measured tRNAval.
We first tested the accumulation of U14 and other C/D snoRNAs derived from independent polycistronic clusters. The U14 probe should detect the four U14 isoforms of the cluster which are nearly identical. A reduction of 70–80% was observed for U14 in atnuf-1 and atnuf-2 compared with wild type plants (Figure 6). Accumulation of snoR22, snoR37 and snoR23 encoded by two duplicated clusters (Barneche et al., 2001) were also drastically reduced in atnuf-1 and atnuf-2 (Figure 6). A similar reduction in both mutants was observed for dicistronic C/D snoRNAs R16 and U43 (Figure 6) and C/D snoRNAs U34a, R19, U36.3, R68 and R40 from six other polycistronic clusters (Figure S3). Complementation of atnuf mutants with AtNUFIP-FLAG-HA gene completely restored normal levels of snoRNAs, confirming that their reduction was specifically due to AtNUFIP gene disruption (Figure S2).
Remarkably, accumulation of snoR80, an H/ACA snoRNA derived from the same cluster encoding C/D snoR37, snoR22 and snoR23 showed a 1.7–2.2-fold increase. This finding indicates that the reduction in C/D snoRNA was not due to reduced accumulation or a maturation defect of the polycistronic precursor in the mutants. This fact was confirmed by semi-quantitative RT-PCR that showed no difference in the snoRNA polycistronic precursor level in the mutants compared with wild type plants (Figure S4).
Few H/ACA snoRNA polycistronic clusters have been identified in Arabidopsis thaliana. One of these clusters corresponds to the dicistronic cluster encoding H/ACA snoR141 and snoR77 (Chen and Wu, 2009). Northern blot analysis showed that these snoRNAs presented an approximately two-fold increase in atnuf mutants (Figure 6).
We then analysed the accumulation of intronic C/D snoRNAs nested in protein coding genes. The snoR24 (Figure 6) and snoR40 (Figure S4) encoded by two different intronic cluster showed both a 90% reduction in atnuf mutants compared with wild type seedlings. A different situation was observed for the single intronic snoR60 (Barneche et al., 2000) which was not affected in the mutants (Figure 6).
We also analysed expression of the monocistronic C/D snoRNAs U3 and U13. Accumulation of both snoRNAs increased slightly in atnuf-1 and atnuf-2 (Figure 6).
Finally, we tested the accumulation of C/D snoR43.1, which is processed from a tRNAGly–snoR43 precursor (Kruszka et al., 2003). Northern blots showed a three- to four-fold enhancement of snoR43.1 in both atnuf mutants (Figure 6).
These results show that atnuf mutants present a strong reduction of C/D snoRNAs derived from polycistronic precursors which represent the majority of C/D snoRNAs in plants. The C/D snoRNAs derived from monocistronic, single intronic or tRNA-snoRNA precursors as well as the H/ACA snoRNAs show increased accumulation in the mutants.
Reduction of C/D snoRNAs correlates with rRNA down-methylation in atnuf mutants
Reduction of methylation guide C/D snoRNAs in atnuf mutants should impair 2′-O-ribose methylation of their target residues. To confirm this finding we analysed the level of methylation of rRNA residues using primer extension assay with limiting dNTP substrate (Barneche et al., 2001). Upon limiting dNTP, the reverse transcriptase becomes sensitive to the 2′-O-ribose methylation and generates a premature stop signal. As a positive control, we assessed the methylation of 25S:Gm2114 directed by snoR60, which is not affected in atnuf mutants. Under limiting dNTP, a premature stop signal appeared at the target rRNA residue in wild type plants (Figure 7a). A similar premature stop signal was detected on rRNA from atnuf-1 and atnuf-2 showing that methylation of this residue was not affected in the mutants (Figure 7a).
We then assessed methylation of 25S:Am2826 directed by snoR24 that is drastically reduced in mutants. Methylation of this residue was clearly detected in wild type plants (Figure 7a). Nevertheless the strong arrest signal under limiting dNTPs was not observed with atnuf-1 or atnuf-2 rRNAs indicating a drastic reduction of methylation of this residue (Figure 7a).
We tested methylation of 25S:Gm1260 and 25S:Gm1275 targeted both by C/D snoR22 with has two guide sequences (Brown et al., 2003b). Methylation of its two targets was revealed by two strong premature stop signals in wild type plants (Figure 7a). The snoR22 levels are reduced in the mutants and there are no premature stops on atnuf-1 and atnuf-2 rRNAs, revealing a drastic reduction of their methylation (Figure 7a).
In atnuf mutants U14 was strongly reduced. Considering that U14 is a conserved C/D snoRNA implicated in pre-rRNA cleavage in animals (Enright et al., 1996) and yeast (Li et al., 1990), we analysed the accumulation of pre-rRNAs as well as rRNAs by real time-qRT-PCR in the mutants (Figure 7b). Specific pair of primers complementary to pre-rRNA spacer sequences (5′ETS and ITS1) were designed (Figure 7b). The analysis showed a 1.5- to two-fold increase for pre-rRNA precursors in atnuf mutants that probably reflects impairment in pre-rRNA processing due to reduction of U14 (Figure 7b). Nevertheless, no impact on steady-state levels of mature rRNA 18S and 25S was observed in atnuf mutants (Figure 7b).
Overall, these results show a significant reduction of the 2′-O-ribose methylation of rRNA residues targeted by C/D snoRNAs encoded by polycistronic clusters in atnuf mutants, with no significant effect on total rRNA accumulation.
Accumulation of C/D scaRNAs is reduced in atnuf mutants
The C/D scaRNPs, which direct modification of snRNAs in Cajal bodies, are related to C/D snoRNPs and contain the 15.5K protein which interacts with NUFIP (Jády and Kiss, 2001; Darzacq et al., 2002). In plants, few scaRNAs have been identified but their genomic organisation has not been clearly defined. We tested whether AtNUFIP is also required for their accumulation.
C/D scaR101, predicted to target methylation of U2 (Marker et al., 2002), is encoded by the opposite strand of At1g20690, a predicted gene with no protein-coding capacity. Clearly scaR101 is expressed in wild type plants and is strongly reduced in atnuf-1 and atnuf-2 mutants (Figure 8a).
C/D scaR102 is predicted to target methylation of U5 (Marker et al., 2002) and was confirmed to be located in plant Cajal bodies (Kim et al., 2010). It was originally described as a monocistronic scaRNA. Nevertheless analysis of flanking genomic sequences reveals a potential C/D scaRX just upstream of scaR102 and a cDNA (AY045928) of 385 nucleotides encompassing both sequences is reported in databanks (Figure 8b). A Northern blot with a scaR102 probe revealed an approximately 160 nucleotide RNA and a longer transcript of approximately 370 nucleotides (Figure 8b). Hybridisation with a probe specific to the scaRX sequence detected only the longer RNA (Figure 8b) revealing that the potential scaRX is not produced in vivo. These data indicate that the 370 nucleotides RNA is a stable transcript that accumulates in vivo and is also a precursor for scaR102 but not for potential scaRX. This finding is reminiscent of some scaRNAs described in animals produced from longer transcripts that also accumulate in vivo (Tycowski et al., 2004). Notably, accumulation of both the scaR102 and the longer transcript was affected in both atnuf-1 and atnuf-2 (Figure 8b). This situation confirms the implication of AtNUFIP in C/D scaR102 assembly and in the stabilisation of the longer transcript.
We assessed whether AtNUFIP is required for accumulation of intronic H/ACA scaR103 predicted to target pseudouridylation of U5 (Chen and Wu, 2009; Kim et al., 2010). No effect was observed in its accumulation in atnuf-1 and atnuf-2 mutants (Figure 8a).
The 15.5K protein is also a core component of U4 snRNP and it was proposed that NUFIP could be implicated in its assembly (Boulon et al., 2008). We tested the accumulation of U4 in the mutants but no effect was observed (Figure 8c).
Reduction of C/D scaRNAs should lead to down-methylation of their target snRNAs. We could not test this hypothesis because the assay requires a large amount of RNA, which could not be obtained from atnuf mutants. Nevertheless Northern blot shows that the accumulation of snRNAs is not affected in atnuf mutants (Figure 8c). This finding suggests that the predicted reduction of snRNA methylation in atnuf mutants does not affect snRNA stability.
- Top of page
- Experimental procedures
- Supporting Information
We have identified the AtNUFIP gene in Arabidopsis and characterized two distinct atnufip mutant lines, revealing that this gene is essential for plant development. AtNUFIP directly interacts with At15.5K and is probably implicated in C/D snoRNP assembly, as revealed by destabilisation of C/D snoRNAs in atnufip mutants. Most remarkably this study reveals that the requirement of AtNUFIP for C/D snoRNP assembly depends on the genomic organisation of C/D snoRNAs. In addition AtNUFIP is also required for the biogenesis of C/D scaRNPs directing methylation of spliceosomal snRNA. These results raise several questions.
AtNUFIP is required for the assembly of snoRNPs and scaRNPs
AtNUFIP has the conserved PEP motif and interacts with At15.5K core C/D snoRNP protein in vitro (Figure 3). This suggests that, similar to NUFIP (McKeegan et al., 2007; Boulon et al., 2008), AtNUFIP has a chaperone function facilitating the assembly of C/D snoRNP. The strong reduction of C/D snoRNAs in atnuf mutants probably results from defective assembly triggering snoRNA degradation (Figure 6). Likewise, the reduction of C/D scaRNAs in atnuf mutants (Figure 8a) is due to impaired scaRNP assembly.
Depletion of NUFIP in HeLa cells leads to an approximate 20% reduction of an H/ACA snoRNA and it was proposed that it also controls assembly of H/ACA snoRNPs (Boulon et al., 2008). In plants the direct implication of AtNUFIP in H/ACA snoRNP assembly seems unlikely considering the moderate increase of all H/ACA snoRNAs in atnufip mutants (discussed below).
The lack of effect of atnuf mutants on U4 is intriguing because the 15.5K protein, which directly interacts with NUFIP, is a core component of the U4 snRNP in eukaryotes. However, a similar result was observed for human or yeast cells depleted of NUFIP or Rsa1 (Boulon et al., 2008).
AtNUFIP and its role in polycistronic C/D snoRNA assembly: a kinetic model
An important observation is that AtNUFIP is not essential for the biogenesis of some C/D snoRNPs. In fact, AtNUFIP becomes critical only for the accumulation of C/D snoRNAs processed from polycistronic precursors, either from independent or intronic clusters, which represent the vast majority of plant snoRNAs.
One simple explanation is that AtNUFIP is required to maintain the normal rate of C/D snoRNP assembly. It has been proposed that any factor affecting the kinetics of RNP assembly triggers the degradation of defective RNA (Doma and Parker, 2007). The specific effect on polycistronic snoRNAs in atnuf mutants would, therefore, be the result of a lower assembly rate of multiple C/D snoRNPs on a single pre-snoRNA, leading to incomplete assembly and rapid degradation of the snoRNA. In this kinetic model, an important factor is the availability of core proteins and other auxiliary factors. Assembly is known to occur on the nascent pre-snoRNAs and is tightly linked to transcription and processing (Ballarino et al., 2005; Darzacq et al., 2006; Hirose et al., 2006). This factor defines a ‘physical and temporal pre-snoRNA nascent space’ in which the assembly and core factors could become limiting on a polycistronic precursor because several complexes have to be assembled concomitantly.
Probably, AtNUFIP also facilitates the assembly on ‘monocistronic’ snoRNAs in vivo but its implication is masked in atnuf mutants. Indeed the monocistronic U3 and U13 snoRNA as well as snoR43 derived from dicistronic tRNA-snoRNA are increased in atnuf mutants (Figure 6). One explanation is that, as snoRNPs derived from polycistronic precursors are not longer assembled, C/D core component or other auxiliary factors would be more available for assembly of the C/D snoRNP on monocistronic units. An alternative explanation is that the massive degradation of polycistronic snoRNAs in atnuf mutants diverts the RNA degradation machinery and this situation would result in a stabilisation of monocistronic C/D snoRNAs. Similar explanations could be invoked for the increase of H/ACA snoRNAs observed in the mutants considering the possibility that common factors could be implicated in the biogenesis of C/D and H/ACA snoRNAs (Rodor et al., 2010).
AtNUFIP sub-cellular localisation and the assembly of snoRNPs in plants
Where does assembly of snoRNP occur? In plants, pre-snoRNA polycistronic precursors have been detected by in situ hybridisation in the Cajal bodies and in the nucleolus (Shaw et al., 1998). Thus, some assembly steps that are linked to pre-snoRNA processing could occur in any, or both, of these compartments in plants. Localisation of the GFP–AtNUFIP fusion in transgenic plants shows diffuse fluorescence in the nucleoplasm, with bright foci in the nucleolar periphery (Figure 4b). This peripheral region could correspond to the granular component of the nucleolus where the last steps of rRNA processing and assembly of ribosomes occurs. The distribution of At15.5K-RFP is different: it is concentrated in the centre of the nucleolus and in Cajal bodies (Figure 4a). GFP–AtNUFIP partially colocalizes with At15.5K-RFP in the nucleolar periphery, suggesting that some steps of snoRNP biogenesis could occur at the nucleolar periphery and even inside the nucleolus in plants.
In animals, the Cajal bodies are assembly factories for both snRNPs and snoRNPs (Narayanan et al., 1999; Verheggen et al., 2002; Qiu et al., 2008). Surprisingly, we never detected AtNUFIP in Cajal bodies in areas where At15.5K-RFP (Figure 4b) and scaRNAs (Kim et al., 2010) are found. This fact is probably due to the very low level of expression of GFP–AtNUFIP in transgenic plants.
AtNUFIP and development
The atnuf mutants are affected at different steps of plant development. The phenotypes are roughly similar in both mutant lines but are much more severe in atnuf-2. Considering that truncated mRNAs are produced in the mutants, this fact could be explained by the expression of a truncated AtNUFIP protein in atnuf-1 (Figure 2). In atnuf-1, the predicted truncated protein would be nearly full length and contain an intact PEP motif, while in atnuf-2 it would be totally ‘disrupted’ and probably degraded.
Developmental defects in atnuf mutants could result from defective ribosomes. The pointed leaf phenotype observed in young atnuf seedlings (Figure 5) is typical of ribosomal protein plant mutants (Van Lijsebettens et al., 1994; Weijers et al., 2001; Degenhardt and Bonham-Smith, 2008).
It is unlikely that defective ribosomes are linked to pre-rRNA processing defects as steady state levels of mature rRNAs are not affected in atnuf mutants (Figure 7b). A similar situation has been reported for the Arabidopsis Atnuc-l1, Atrtl2 and xrn2 mutants, which are affected in pre-RNA processing but have no effect on steady-state levels of mature rRNAs (Kojima et al., 2007; Comella et al., 2008; Zakrzewska-Placzek et al., 2010).
More probably defective ribosomes in atnuf mutants could be due to down-methylation of rRNAs. In Arabidopsis, most C/D snoRNAs are encoded by polycistronic C/D snoRNA genes and their reduction in atnuf mutants correlates with concomitant reduction of the methylation of their target rRNA residues. Thus, atnuf mutants should present an important reduction in the global level of rRNA methylation and this could reduce the translational capacity of the ribosomes (Esguerra et al., 2008).
Developmental defects of atnuf mutants could also be due to deficient splicing. The atnuf mutants show a drastic reduction of C/D scaR101 and scaR102. They target respectively methylation of U2:Cm29, which is specific to plants and drosophila (Huang et al., 2005), and U5:Gm41, conserved in many species (Massenet et al., 1998). In Xenopus oocytes and human cells, depletion of U2 modifications affects the formation of spliceosomes and has an impact on splicing (Yu et al., 1998; Dönmez et al., 2004; Zhao and Yu, 2004). Interestingly, splicing of AtNUFIP truncated mRNA is affected in the atnuf mutants (Figure 2c). At present, except for the example of U2, little is known on the effect of snRNA modification on splicing. The atnuf mutants now provide the opportunity to address this question using a transcriptomic/sequencing approach.
Future prospectives in AtNUFIP function
Ribosome and splicing defects probably both contribute to mutant phenotypes, but cannot completely explain the strong developmental defects of atnuf mutants. Indeed, whereas the level of reduction of C/D snoRNA and scaRNAs are comparable in both mutant lines, the developmental phenotypes are much more severe in atnuf-2. We suspect that AtNUFIP has additional unidentified RNA substrates and functions. These should direct the study of AtNUFIP in the future.
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- Experimental procedures
- Supporting Information
Plant materials and histology studies
Arabidopsis are from the Columbia 0 ecotype. Mutant atnuf-1 (SALK-134962) and atnuf-2 (GABI-680F01) lines were obtained from the European Arabidopsis Stock Centre (http://arabidopsis.info) and Gabi-Kat (http://www.gabi-kat.de) respectively. Plant cultures were done on MS medium under continuous light for in vitro culture and in soil under a 16:8 h light/dark cycle.
Longitudinal sections of inflorescence meristems and pistils were performed as described previously (Carles et al., 2010).
RNA extraction, RT-PCR, qPCR and RLM RACE
Total RNAs were extracted from tissues by the guanidine hydrochloride method (Logeman et al., 1987). For RNA extraction from siliques, the Invisorb spin plant RNA mini kit (Invitek, http://www.invitek.eu) was used. RNA samples were then treated with RQ1 RNase-free DNase (Promega, http://www.promega.com). For PCR substrates, cDNA was produced using the StrataScript First Strand Synthesis System (Stratagene, http://www.genomics.agilent.com). RLM RACE to map AtNUFIP mRNA extremities used the FirstChoice RLM RACE kit (Ambion, http://www.ambion.com), with total RNA from 15-day-old seedlings.
For real-time PCR, cDNAs were obtained from RNAs extracted from 14 to 18-day-old seedlings using random primers. Real-time PCR was performed using Light Cycler 480 SYBR Green I Master (Roche, http://www.roche.com) using the following parameters: start cycle: 95°C for 10 min; amplification: 45 cycles of 95°C × 10 sec, 65°C × 10 sec, 72°C × 20 sec; cycle for melting curve: 95°C × 5 min, 65°C × 1 min, increase to 97°C with a slope of 0.11 sec; cooling at 40°C for 30 sec, then stop. Amplification was performed in triplicate for three independent biological samples. Actin and GAPA genes were used as an internal standardisation controls.
Total RNAs from 14 to 18-day-old seedlings were separated on a 7% acrylamide–8.3M urea gel, transferred to membrane (Hybond N+; Amersham, http://www.gelifesciences.com) and UV light crosslinked. Membranes were incubated overnight with [32P]-5′end-labelled oligonucleotide probe at 37°C in 6× SSC, 5× DENHARDT’S, 1% SDS, 100 μg ml−1 salmon sperm DNA. Membranes were washed 15 min at 37°C and 15 min at 42°C in 6× SSC, 1% SDS then twice at 42°C in 1× SSC and 1% SDS. After exposure, membranes were scanned with a STORM 860 (Molecular Dynamics, http://www.moleculardynamics.com) and signals quantified in comparison to tRNAVal.
AtNUFIP coding sequence was amplified from the pENTRY221-At5g18440 clone (ABRC) and At15.5K-3 (At4g22380) from a seedling cDNA library. The GFP::AtNUFIP and At15–5K::RFP were obtained using the Gateway system (Invitrogen, http://www.invitrogen.com). For complementation of mutants, the AtNUFIP genomic sequence from 1 kb upstream of the transcription initiation site to the end of the coding sequence were cloned in frame into a 2×FLAG::2×HA-pCAMBIA 1300 vector.
Creation of transgenic lines expressing fluorescent proteins and localization
Stable transgenic plants expressing the AtNUFIP and At15.5K fluorescence fusions were produced by Agrobacterium-mediated transformation using the floral dip method (Walkerpeach and Velten, 1994). Plants expressing U2B′′-GFP (Collier et al., 2006) were provided by Dr P. Shaw (John Innes Institute, Norwich).
Complementation of atnuf mutants with AtNUFIP-FLAG-HA
Atnuf-1 and atnuf-2 heterozygous mutants were transformed with promAtNUFIP::AtNUFIP-FLAG-HA construct using Agrobacterium-mediated floral dip method. The stable insertion of the AtNUFIP-FLAG-HA transgene was confirmed by PCR genotyping. Accumulation of AtNUFIP-FLAG–HA protein was confirmed by Western blot using anti-HA-HRP antibody (Sigma, http://www.sigmaaldrich.com).
Yeast two-hybrid assay
AtNUFIP and At15.5K-3 (At4g22380) ORFs were cloned in frame with the activator or binding Gal4 domains in pGAD.T7 and pGBK.T7 vectors (Clontech, http://www.clontech.com). The Saccharomyces cerevisiae strain YRG2 (Stratagene), carrying a Gal4-based yeast two-hybrid reporter system, was transformed by lithium chloride treatment (Gietz et al., 1992). Yeast cells were plated on minimal YNB medium with required amino acids with (control) or without (Y2H assay) histidine and grown at 30°C for 6 days.
GST pull down assay
GST-At15.5K-3 and GST proteins were produced in Escherichia coli BL21 and purified using the BULK GST Purification Modules kit (GE Healthcare, http://www.gehealthcare.com). [35S] Met-AtNUFIP was synthesized with the PROMEGA TNT Coupled Reticulocyte Lysate System. Binding was performed with 10 μg of GST-At15.5K-3 or GST in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 0.1 mm EDTA, 0.1% NP-40, 2 mm DTT, 1 mm PMSF, 2 mm benzamidine, 1 μg ml−1 leupeptin, 1 μg ml−1 pepstatine and 10% glycerol. After three washes using the same buffer, bound proteins were eluted in Laemmli buffer and separated by SDS-PAGE. Labelled proteins were visualized by a Storm 860 (Molecular Dynamics).
Mapping 2′-O-ribose methylation on rRNAs
To map 2′-O-ribose methylation, primer extension was performed on 1.5 μg of total RNA with 5′end-labeled oligonucleotides and two concentrations of dNTPs (1 mm or 0.004 mm). The sequencing ladder was performed with the same primer on the corresponding DNA sequence as template.
List of oligonucleotides
All primer and oligonucleotide probes are given in Table S1.
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- Experimental procedures
- Supporting Information
We would like to thank Dr E. Bertrand and Dr. J. Saez-Vasquez for helpful discussions, Dr R. Cooke for correcting the English, Dr Peter Shaw for providing us the 35S:U2B′′–GFP Arabidopsis seeds and Dr D. Pontier for the gift of the FLAG-HA pCAMBIA 1300 vector. We are also grateful to Dr H. Murakami and the Global COE program from Nagoya University for supporting J.R. stage in Japan. This work was supported by a grant to J.R. from the Ministère de l’Enseignement et de la Recherche and the Project CNRS-JSPS 2008–2009, no. PRC449.
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- Experimental procedures
- Supporting Information
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- Experimental procedures
- Supporting Information
Figure S1. Alignment of At15.5K proteins and RT-PCR analysis of expression of At15.5K genes. (A) Alignment of the three Arabidopsis 15.5K proteins At15.5-1 (At5g20160) At15.5K (At4g12600) and At15.5K-3 (At4g22380) with the human 15.5K and the yeast Snu13p sequences. (B) Expression of the At15.5K mRNAs detected by semi-quantitative RT-PCR, using primers that distinguish the three mRNAs.
Figure S2. Complementation of atnuf-1 and atnuf-2 mutants with tagged AtNUFIP-FLAG–HA. atnuf-1 and atnuf2 homozygous lines were complemented with the AtNUFIP-FLAG–HA tagged coding sequence including all introns and driven by the AtNUFIP promoter (see Experimental procedures). (a) and (b) indicate different complementation lines of the same mutant. (A) Western blot detecting AtNUFIP-FLAG-HA in the complemented lines using anti-HA antibody. (B) Northern blot of snoRNAs and scaRNAs in wild type, atnuf mutants and complemented lines. (C) Phenotypic analysis of atnuf-1, atnuf-2 and complemented lines, compared with wild type plants.
Figure S3. Northern blot analysis of additional polycistronic snoRNAs in atnuf mutants. The genomic organisation of the distinct snoRNAs is shown. The transcription initiation site is indicated by angled arrow. Grey and black arrows correspond to C/D snoRNAs and H/ACA snoRNAs respectively.
Figure S4. Semi-quantitative RT-PCR analysis of polycistronic snoRNA precursor in atnuf mutants. Amplification was carried out using different pair of primers designed to detect accumulation of snoRNA precursor. The snoRNA genomic organization and primer positions are presented. In the case of the clusters including R37, R22 and R80, three distinct bands are detected because this cluster is encoded by three distinct loci which are all amplified by the primers. gDNA represents PCR amplification of genomic DNA as a control of primer specificity. Controls for RT-PCR were made in the absence of reverse transcriptase as indicated.
Table S1. Primers used for the RT-PCR and RLM-RACE analysis and oligonucleotides used for northern blot hybridization.
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