Molecular cloning and targeting of a fibrillarin homolog from Arabidopsis

Abstract

Fibrillarin is a nucleolar protein known to be involved in the processing of ribosomal RNA precursors. We isolated AtFbr1, a cDNA encoding a homolog of fibrillarin in Arabidopsis. The cDNA is 1.2 kb in size and encodes a polypeptide of 310 amino acid residues with a molecular mass of 33 kD. AtFbr1 is expressed at high levels in the flower and root tissue and at a slightly lower level in leaf tissue, whereas it was nearly undetectable in siliques. Expression of AtFbr1 was compared with that of the FLP (fibrillarin-like protein) gene identified by the Arabidopsis genome project. Abscisic acid treatment resulted in the down-regulation of the expression of both AtFbr1 and FLP genes in seedlings, although the degree of suppression was higher for FLP than for AtFbr1. In addition, the expression level of FLP decreased with the age of the seedlings, whereas AtFbr1 did not exhibit any detectable change. The subcellular localization of AtFbrl was studied with an in vivo targeting approach using a fusion protein, and was found to be correctly targeted to the nucleolus in protoplasts when expressed as a green fluorescent fusion protein (GFP). Deletion experiments showed that the N-terminal glycine- and arginine-rich region is necessary and sufficient to target AtFbr1 to the nucleolus.

Figure 1

Nucleotide and deduced amino acid sequence of the fibrillarin cDNA from Arabidopsis (AtFbrl). Boxed sequences denote the target sequences for the oligonucleotides used in the PCR cloning procedure. The primers used in the construction of various deletion mutants are indicated by arrows. The consensus sequences of the GAR domain are underlined. The nucleotide sequence of the AtFbrl cDNA was deposited in GenBank with accession no. AF187871.

Figure 2

Alignment of the amino acid sequence of AtFbrl with other fibrillarin homologs. The deduced amino acid sequence AtFbrl was aligned with sequences obtained from public databases and refer to the following: Fbrl-Arabidopsis, Arabidopsis fibrillarin-like protein (accession no. CAB43694); Fbrl-human, human fibrillarin (accession no. A38712); Fbrl-X.laevis, Xenopus fibrillarin (accession no. P22232); and Fbrl-yeast, yeast fibrillarin (accession no. P15646). Gaps represented by dashes were introduced to produce the best match among the five species. Identical amino acid residues between AtFbrl and other homologs are shown in uppercase letters. Asterisks mark the RNA-binding consensus motif.

Figure 3

Southern-blot analysis of Arabidopsis genomic DNA. Five micrograms of genomic DNA digested with the indicated restriction enzymes was electrophoresed and transferred to a nylon membrane. Hybridizations were carried out with a 150-bp fragment from the 3′-non-coding region of AtFbrl (A) or FLP (B).

Figure 4

Organ-specific expression of two fibrillarin genes. Total RNA (15 μg) isolated from various tissues was size-fractionated in a formaldehyde-agarose gel and transferred onto a nylon membrane. Specific probes of AtFbrl and FLP were used as the hybridization probes. 18S rRNA was used to monitor the loading of the RNA samples. F, Flowers; L, leaves; R, roots; and S, siliques.

Figure 5

Induction of AtFbrl and FLP gene expression. A, Arabidopsis seedlings grown in MS liquid medium were treated with 100 μm abscisic acid for the indicated periods of time. B, Arabidopsis seedlings were planted on MS plates and harvested after the indicated number of days. Total RNA (15 μg) was analyzed by northern-blot analysis using the specific probes for AtFbrl and FLP. 18S rRNA was used to monitor equal loading of RNA samples.

Figure 6

Targeting of AtFbr1 to the nucleolus. A, Schematic representation of the fusion constructs. The constructs AtFbr1::smGFP and NLS::smGFP contain the full-length AtFbr1 and SV40 nuclear localization signal, respectively. B, In vivo targeting of the GFP fusion proteins in protoplasts. The fusion constructs were introduced into Arabidopsis protoplasts prepared from whole seedlings grown in liquid MS medium for 1 to 2 weeks. The protoplasts were incubated at 22°C in the dark for 12 to 48 h. The panels AtFbrl::smGFP and NLS::smGFP show the fluorescence of the constructs transformed into protoplasts. The protoplast population contains protoplasts originated from root cells. Note that the red fluorescent signal in some protoplasts is chlorophyll. The panel AtFbr1::smGFP + EtBr shows protoplasts stained with EtBr (5 μg/mL) after transformation.

Figure 7

Targeting of smGFP fused to various deletion mutants of AtFbrl. A, Schematic representation of the deletion constructs. B, Protoplasts transformed with the various deletion constructs and their fluorescent signals observed through a fluorescence microscope.