UBA1 and UBA2, two proteins that interact with UBP1, a multifunctional effector of pre-mRNA maturation in plants

Abstract

Nicotiana plumbaginifolia UBP1 is an hnRNP-like protein associated with the poly(A)(+) RNA in the cell nucleus. Consistent with a role in pre-mRNA processing, overexpression of UBP1 in N. plumabaginifolia protoplasts enhances the splicing of suboptimal introns and increases the steady-state levels of reporter mRNAs, even intronless ones. The latter effect of UBP1 is promoter specific and appears to be due to UBP1 binding to the 3' untranslated region (3'-UTR) and protecting the mRNA from exonucleolytic degradation (M. H. L. Lambermon, G. G. Simpson, D. A. Kirk, M. Hemmings-Mieszczak, U. Klahre, and W. Filipowicz, EMBO J. 19:1638-1649, 2000). To gain more insight into UBP1 function in pre-mRNA maturation, we characterized proteins interacting with N. plumbaginifolia UBP1 and one of its Arabidopsis thaliana counterparts, AtUBP1b, by using yeast two-hybrid screens and in vitro pull-down assays. Two proteins, UBP1-associated proteins 1a and 2a (UBA1a and UBA2a, respectively), were identified in A. thaliana. They are members of two novel families of plant-specific proteins containing RNA recognition motif-type RNA-binding domains. UBA1a and UBA2a are nuclear proteins, and their recombinant forms bind RNA with a specificity for oligouridylates in vitro. As with UBP1, transient overexpression of UBA1a in protoplasts increases the steady-state levels of reporter mRNAs in a promoter-dependent manner. Similarly, overexpression of UBA2a increases the levels of reporter mRNAs, but this effect is promoter independent. Unlike UBP1, neither UBA1a nor UBA2a stimulates pre-mRNA splicing. These and other data suggest that UBP1, UBA1a, and UBA2a may act as components of a complex recognizing U-rich sequences in plant 3'-UTRs and contributing to the stabilization of mRNAs in the nucleus.

FIG. 1.

Sequence alignment (A) and schematic structure (B) of the UBA1 proteins. Alignment was generated by using the CLUSTAL W program (59) with the default parameters and was improved manually. Conserved amino acids were shaded by using the BOXSHADE program available at http://www.ch.embnet.org/software/BOX_form.html. Amino acids identical or similar in two of the three analyzed sequences have black and gray backgrounds, respectively. Dashes indicate gaps. The RNP2 sequence (six amino acids) and the RNP1 sequence (eight amino acids) of the RRM are overlined. Genomic sequences encoding UBA1a (GenBank accession number AC007232; protein identification: AAD25815), UBA1b (GenBank accession number AC007232; protein identification: AAD25814), and UBA1c (GenBank accession number AC003058; protein identification: AAC16468) have recently been deposited in GenBank. The predicted size of the UBA1c protein is 849 amino acids. In the alignment, we included only the middle part of the predicted UBA1c protein, which is homologous to UBA1a and UBA1b. K/S/D/E, domain rich in lysine, serine, and aspartic and glutamic acid residues; K/E, domain rich in lysine and glutamic acid residues. Open ovals represent protein regions without any obvious characteristic features.

FIG. 2.

Sequence alignment (A) and schematic structure (B) of the AtUBA2 proteins. The alignment also includes 120 amino acids of UBA1a (RRM plus 39 amino acids upstream of the RRM) which share strong sequence similarity with the UBA2 RRM1, including the corresponding upstream region. Genomic sequences encoding UBA2a (GenBank accession number T44669; protein identification: CAC00749), UBA2b (GenBank accession number AC004261; protein identification: AAD12005), and UBA2c (GenBank accession number AP000370; protein identification: BAA97064) have recently been deposited in GenBank. Which of the 5′-proximal AUG codons in UBA2c is used for translation initiation is not clear. However, the first methionine codon has the best initiation consensus sequence. The nucleotide sequence of the relevant region is CTGTAACCATGGATATGATG. D/E, domain rich in aspartate and glutamate residues; G, domain rich in glycine residues. For other details, see the legend to Fig. 1.

FIG. 3.

Reconstitution of the yeast two-hybrid interactions. Combinations of the bait and prey plasmids (indicated on both panels) were transformed into the yeast reporter strain, and the cells were grown on a synthetic medium lacking tryptophan and leucine (SD-TL) or histidine, tryptophan, and leucine (SD-HTL). Positive interactions obtained on SD-HTL medium were verified by β-galactosidase assays (data not shown). Interactions between UBA2a and UBP1 (from both N. plumbaginifolia and A. thaliana) were observed only when UBA2a and UBP1 were expressed from pGAD424 and pGBT9, respectively, and not in the reciprocal combination. ND, not determined.

FIG. 4.

Protein interactions studied by in vitro pull-down assays. GST-UBA1a and GST-UBA2a wild-type or mutant fusion proteins and GST alone were overexpressed in E. coli and immobilized on glutathione-Sepharose 4B beads. The beads were incubated with RNase-treated whole-cell protein extracts prepared from nontransfected N. plumbaginifolia protoplasts (lanes N) or protoplasts transfected (lanes T) with pUBP1 (encoding NpUBP1) (A), pAtUBP1b-HA (B), pUBA1a-HA (C), or pUBA2a-HA (D). (A and B) Pull-down assays with GST, GST-UBA1a, and GST-UBA2a. (C and D) Extracts were incubated with glutathione beads with immobilized GST-UBA2a or GST-UBA1a. (E and F) UBP1, UBA1a, and UBA2a interactions are not RNA mediated. Protein extracts from protoplasts overexpressing the proteins indicated were incubated with glutathione beads coated with GST, GST-UBA1a, GST-UBA1aM, GST-UBA2a, or GST-UBA2aM. Proteins retained on the beads were analyzed by SDS-PAGE and subsequent Western blotting with UBP1 MAb DG6 (A and E) and anti-HA MAb 3F10 (B, C, D, and F).

FIG. 5.

Subcellular localization of UBA1a and UBA2a by cellular fractionation (A and B) or imaging of protoplasts expressing GFP fusions (C). (A) N. plumbaginifolia protoplasts were transfected with the empty vector pDEDH-HA or plasmids pUBP1-HA and pUBA1a-HA. (B) Protoplasts were transfected with pUBA2a-HA. Cell extracts were fractionated as described before (34). Lanes N, C, and T, nuclear, cytoplasmic, and total cellular protein fractions, respectively. Proteins were resolved by SDS-PAGE and analyzed by Western blotting with mouse anti-HA MAb 12CA5 (A), anti-U2B" MAb 4G3 (A and B, lower panel), or rat anti-HA antibody 3F10 (B, upper panel). The protein marked with an asterisk cross-reacts nonspecifically with the anti-U2B" MAb 4G3. The 40-kDa nuclear protein, marked with a double asterisk, cross-reacts nonspecifically with the anti-HA MAb 12CA5. (C) GFP imaging of protoplasts transfected with pUBA1a-GFP, pUBA2a-GFP, and pDEDH-GFP (expressing GFP alone). Protoplasts were analyzed by using a Zeiss Axioplan epifluorescence microscope. The arrow indicates an untransfected protoplast. Bars, 50 μm.

FIG. 6.

Determination of RNA-binding specificities of UBA1a and UBA2a. (A) Coomassie blue-stained gel of overexpressed GST-UBA1a and GST-UBA2a proteins purified on glutathione-Sepharose 4B. Each lane was loaded with 1.5 μg of protein. Positions of molecular mass markers (Amersham Pharmacia Biotech) are indicated on the left (lane M). (B and C) Competition of homoribopolymers with UV cross-linking of the ³²P-labeled CaMV 3′-UTR RNA to recombinant GST-UBA1a (B) and GST-UBA2a (C). Polymers poly(C), poly(G), poly(A), and poly(U), indicated at the top of each panel, were added at 2-fold (lanes 2, 6, 11, and 15), 5-fold (lanes 3, 7, 12, and 16), 25-fold (4, 8, 13, and 17), and 100-fold (lanes 5, 9, 14, and 18) excesses (calculated in moles of nucleotides) over labeled RNA. Lanes 1 and 10, cross-linking with no competitor added. Only relevant gel fragments are shown. The approximately 35-kDa abundant polypeptide present in the GST-UBA1a preparation, most likely corresponding to the GST portion of the fusion protein, does not cross-link to RNA.

FIG. 7.

Effects of overexpression of UBA1a and UBA2a on the accumulation of reporter RNAs transiently expressed in N. plumbaginifolia protoplasts. (A) Effect of UBA1a overexpression on the accumulation of the intronless Syn3 reporter RNA. Protoplasts were cotransfected with 5 μg of the reporter plasmid and with empty vector alone (lanes 2 and 8), with increasing amounts of pUBA1a-HA (3, 10, and 15 μg; lanes 3 to 5 and 9 to 11), or with increasing amounts of pUBP1-HA (3 and 10 μg; lanes 6, 7, 12, and 13). Lanes 2 to 7, Syn3 RNA transcribed from the CaMV promoter; lanes 8 to 13, Syn3 RNA transcribed from the GLB promoter. The total amount of plasmid was kept constant (20 μg) by the addition of appropriate amounts of empty expression vector pDEDH/Nco. Mappings with the U2 probe, which detects a 50-nucleotide-long 5′-terminal fragment of endogenous U2 RNA, were performed in the same reactions; U2-protected bands are indicated. Lane 1, aliquot of undigested probes. Size markers (in nucleotides) are indicated on the left. Relative RNA yields are indicated in the lanes. Diagrams of relevant portions of plasmids expressing Syn3 and GUS reporter RNAs from CaMV and GLB promoters are shown below the gel. Horizontal arrows indicate transcription start sites, and vertical arrows indicate polyadenylation sites. (B) Summary of the effect of UBA1a on the abundance of different reporter RNAs initiated at the CaMV or GLB promoter. Values for expression from pSyn3 are means and standard deviations of three (plasmid concentrations, 3 and 15 μg) or four (plasmid concentration, 10 μg) independent transfection experiments. Remaining values are means of two independent transfection experiments. Quantification is based on PhosphorImager data corrected for endogenous U2 snRNA recovery. (C) Effect of overexpression of UBA2a on the accumulation of the intronless Syn3 reporter RNA expressed in N. plumbaginifolia protoplasts. Protoplasts were cotransfected with 5 μg of the reporter plasmid and with empty vector alone (lanes 2, 6, 8, and 12), with increasing amounts of pUBA2a-HA (1, 3, and 10 μg; lanes 3 to 5 and 9 to 11), or with 10 μg of pUBP1-HA (lanes 7 and 13). Lanes 2 to 7, Syn3 RNA transcribed from the CaMV promoter; lanes 8 to 13, Syn3 RNA transcribed from the GLB promoter. The total amount of plasmid was kept constant (15 μg) by the addition of appropriate amounts of empty expression vector pDEDH/Nco. Mappings with the U2 probe, which detects a 50-nucleotide-long 5′-terminal fragment of the endogenous U2 RNA, were performed in the same reactions. Relative RNA yields are indicated in the lanes. Size markers (in nucleotides) are indicated on the left (lane 1). Lane 14, aliquot of undigested probes. In an additional experiment with 3, 10, and 15 μg of plasmid expressing UBA2a-HA, the respective yields for Syn3 RNA transcribed from the CaMV promoter were 3.4, 5.6, and 7.4, and those for Syn3 RNA transcribed from the GLB promoter were 1.3, 1.1, and 2.8.

FIG. 8.

Effects of UBA1a, UBA2a, and UBP1 on the accumulation of Syn3 reporter RNA, as analyzed on a high-resolution polyacrylamide gel. RNA from protoplasts cotransfected with 5 μg of pSyn3 and either 10 μg of empty vector pDEDH-HA (lanes 2, 3, 4, and 5), pUBP1 (lanes 6, 7, 8, and 9), pUBA1a-HA (lanes 11, 12, 13, and 14), or pUBA2a-HA (lanes 15, 16, 17, and 18) was resolved on a 6% polyacrylamide-8 M urea gel and analyzed by Northern blotting. Antisense Syn3 RNA was used as a probe. Lanes 1 and 10, RNA from nontransfected protoplasts; lanes 2, 6, 11, and 15, total RNA; lanes 3, 7, 12, and 16, RNA treated with RNase H and oligo(dT); lanes 4, 8, 13, and 17, poly(A)⁺ RNA; lanes 5, 9, 14, and 18, poly(A)⁻ RNA. Positions of size markers (in nucleotides) are indicated on the right. Only the relevant portion of the gel is shown.