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First published online 29 June 2004
doi: 10.1242/jcs.01194

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Multiple domains control the subcellular localization and activity of ETR-3, a regulator of nuclear and cytoplasmic RNA processing events

Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

View larger version (64K): [in a new window]   Fig. 1. Cloning of chicken ETR-3. (A) Chicken ETR-3 sequence. In human and mouse the ETR-3 gene has at least five different first exons encoding three different N termini (Li et al., 2001) (and unpublished observations). The open reading frame of chicken ETR-3 was amplified using primers containing the termination codon and the furthest downstream start codon. RRMs are boxed, with RNP sequences in bold. Leucine-rich regions in the divergent domain are highlighted in gray, and an arginine/lysine-rich region at the C terminus is in italics. Residue numbers shown to the right are based on variant L sequence; the four amino acids unique to variant 4 (see below) fall between residues 335 and 336 of variant L and are underlined. (B) Two full-length isoforms of ETR-3 generated by alternative splicing were amplified by RT-PCR from embryonic chicken heart RNA. Variant 4 is identical to variant L except for use of an alternative 5' splice site in an exon corresponding to human and mouse exon 10, leading to the insertion of four residues (TVNS) in the divergent domain. Exons are numbered based on comparisons to human and mouse genes. The probe used in D is shown. (C) Chicken ETR-3 variants were cloned into pcDNA3.1(+) expression vectors to express untagged proteins and transfected into quail QT35 fibroblasts. Transfected samples (4 and L) were compared to untransfected fibroblasts (U) and embryonic day 12 chicken heart (d12) by western blot with a rabbit polyclonal antibody against ETR-3. Both variants 4 and L comigrate at their predicted size (52 kDa) with full-length ETR-3 in chicken heart. (D) RNase protection assays demonstrate that variant L is the predominant isoform expressed throughout heart development. P, undigested probe diagrammed as shown diagrammatically in B; Y, yeast RNA; d8, embryonic day 8 chicken heart RNA; A, adult chicken heart RNA. Arrowheads indicate the expected locations of the undigested probe and protected fragments for each isoform (4, variant 4 protected fragment; L, variant L protected fragment). The expected sizes for the probe and protected fragments are shown in parentheses. The two faint bands between the variant 4 and variant L protected fragment sizes are background bands, as they appear in all lanes including the yeast RNA control.

View larger version (56K): [in a new window]   Fig. 2. Identification of a C-terminal NLS. (A) GFP was fused to the N terminus of full-length chicken ETR-3 variant L (GFPcETR3vL) and deletion constructs were subsequently made removing the last 58 (427-484) or 20 (465-484) amino acids from the C terminus, producing truncated proteins with molecular masses of 72.5 kDa or 76.6 kDa (GFPcETR3 and GFPcETR3, respectively). These C-terminal fragments were also fused to the C terminus of a GFP-PK fusion construct to produce chimeric proteins with molecular weights of 81.8 kDa (GFP-PK-) or 77.7 kDa (GFP-PK-). (B) GFPcETR3vL is nuclear. The C-terminal deletion constructs GFPcETR3 and GFPcETR3, in contrast, are predominantly cytoplasmic. The localization of GFP-PK is shifted to the nucleus by the addition of the C-terminal 58 or 20 amino acids of ETR-3 (GFP-PK- and GFP-PK-, respectively). Scale bar: 10 µm.

View larger version (81K): [in a new window]   Fig. 3. Identification of nuclear localization and export elements in the divergent domain. (A) The divergent domain of ETR-3 variant L was divided into four parts (residues 188-240, 241-293, 294-346, and 347-399) and deletions of each of these four segments were made within GFPcETR3vL (GFPDD.1-4) and GFPcETR3 (GFPDD.1-4). (B) Western blot analysis using an anti-GFP antibody confirmed that all expression plasmids expressed proteins of the expected sizes, and the deletion mutants all expressed protein at comparable or greater levels than the full-length protein. (C) Deletion of the fourth divergent domain region alone (GFPDD.4) resulted in loss of nuclear localization in transfected cells, indicating this region is required for nuclear localization. Deletion of the first and second divergent domain regions in conjunction with the C terminus (GFPDD.1 and GFPDD.2) resulted in partial restoration of nuclear localization, indicating the presence of sequences required for cytoplasmic localization. Scale bar: 10 µm.

View larger version (39K): [in a new window]   Fig. 4. A region of the divergent domain is sufficient for cytoplasmic localization in primary embryonic chicken cardiomyocytes. (A) The first three quadrants of the divergent domain (residues 188-346) of ETR-3 variant L were fused to the NLS-GFP-PK chimeric protein (NLS-GFP-PK-DD.123). (B) Western blot analysis using an anti-GFP antibody confirmed that proteins of the expected sizes were produced from both expression plasmids. (C) The NLS-GFP-PK chimeric protein is normally nuclear (top panels). Addition of the first three quadrants of the divergent domain resulted in cytoplasmic localization in transfected primary embryonic chicken cardiomyocytes (bottom panels). Scale bar: 10 µm.

View larger version (36K): [in a new window]   Fig. 5. Nuclear localization does not restore full splicing activity in the absence of the C terminus. (A) Incorporation of the SV40 large T antigen NLS at the C-termini of the GFPcETR3 and GFPDD.4 fusion proteins (GFPcETR3NLS and GFPDD.4NLS, respectively) conferred nuclear localization in COS-M6 cells. Scale bar: 10 µm. (B) RT-PCR analysis was performed (upper panel) to determine the extent of exon inclusion (lower panel) in cotransfection assays with a cTNT minigene. While all of the GFP-ETR3 fusion constructs gave levels of exon inclusion that were statistically different from that of the minigene alone (P0.05), GFPcETR3 and GFPDD.4 activated exon inclusion only slightly above the basal level. The activity of the C-terminal deletion mutant was not enhanced by the addition of an NLS, as GFPcETR3NLS did not activate levels of exon inclusion significantly higher than those observed for GFPcETR3. When the divergent domain deletion was restored to the nucleus, however, GFPDD.4NLS enhanced exon inclusion significantly above the level observed for GFPDD.4 and close to the level observed for the full-length protein. The average percentages of total mRNAs containing the alternative exon plus or minus the standard error of the mean are shown for each (n4).

View larger version (104K): [in a new window]   Fig. 6. Nuclear export of ETR-3 is leptomycin B (LMB)-sensitive. GFPcETR3 is cytoplasmic in the absence of LMB in untreated (upper right panel) or mock-treated cells (data not shown), but accumulates in the nucleus following one hour of exposure to 1 µM LMB similar to a positive control containing known NLS and LMB-sensitive NES elements (GFP-PK-Vpr). In three experiments the nuclear concentration of GFPcETR3 increased in the presence of LMB, but some GFPcETR3 was retained in the cytoplasm (this result is shown). In a fourth experiment, LMB treatment resulted in an exclusively nuclear localization of GFPcETR3 (data not shown). The extent of nuclear accumulation of GFPcETR3 in each case is comparable to GFP-PK-Vpr. A cytoplasmic fusion protein that does not enter the nucleus (GFP-PK) is not affected by LMB treatment. No differences in viability were observed in LMB-treated versus untreated or mock-treated cells. Scale bar: 10 µm.

View larger version (63K): [in a new window]   Fig. 7. Identification of regions important for cytoplasmic localization in RRM1 and RRM2. (A) Deletions within the first two RRMs of ETR-3 (residues 1-89 and 90-178, respectively) were made within GFPcETR3vL (GFPRRM.1-2) and GFPcETR3 (GFPRRM.1-2), producing truncated proteins with molecular masses of 68.9 kDa (GFPRRM.1), 69.0 kDa (GFPRRM.2), 62.4 kDa (GFPRRM.1), and 62.6 kDa (GFPRRM.2). (B) Deletions in RRM1 and RRM2 alone had no effect on localization, but deletions made in conjunction with the deletion of the NLS at the C terminus resulted in partial nuclear localization, indicating sequences within the first two RRMs are important for cytoplasmic localization of ETR-3. Scale bar: 10 µm.

View larger version (11K): [in a new window]   Fig. 8. Functional domains important for localization and splicing activity of chicken ETR-3. Deletion and gain-of-function analyses revealed an NLS within the last 20 amino acids at the C terminus and CRM1-dependent nuclear export activity within the divergent domain. A second region involved in nuclear localization in the last 53 amino acids of the divergent domain (dotted line) was identified by deletional analysis. Regions within the first two RRMs were also identified by deletional analysis that are important for cytoplasmic localization, though this is perhaps due to the RNA binding capacity of these domains. The first quadrant of the divergent domain and the C terminus were also found to be important for splicing activation by ETR-3 independent of its localization to the nuclear compartment.

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