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. 2005 Jun;46(6):1882-90.
doi: 10.1167/iovs.04-1286.

Identification of novel murine- and human-specific RPGRIP1 splice variants with distinct expression profiles and subcellular localization

Xinrong Lu  1 , Paulo A Ferreira
Affiliations

Identification of novel murine- and human-specific RPGRIP1 splice variants with distinct expression profiles and subcellular localization

Xinrong Lu et al. Invest Ophthalmol Vis Sci. 2005 Jun.

Abstract

Purpose: Mutations in RPGRIP1 cause Leber congenital amaurosis. The human and bovine RPGRIP1 undergo alternative splicing. A single murine rpgrip1 transcript has been reported, but distinct expression profiles of RPGRIP1 isoforms exist between species in the retina. To elucidate the heterogeneity of RPGRIP1 isoforms and the degree of functional redundancy among these, we extended the analysis of RPGRIP1 to the region between exons 12 and 14, which undergoes significant alternative splicing.

Methods: Identification of alternative splice transcripts of murine and human RPGRIP1 was performed by reverse transcription-polymerase chain reaction (RT-PCR). The murine rpgrip1 isoforms were analyzed by immunoblot and immunocytochemistry analysis of murine retinas and transient transfected cultured cells.

Results: A novel murine-specific transcript, rpgrip1b was identified. It arises from the extension of exon 13, leading to the premature truncation of rpgrip1 and deletion of its C2 and RID domains. It is predominantly expressed in the retina, where it is more abundant than the transcript(s) encompassing the constitutive exons 12 to 14. Conversely, the human retina lacks rpgrip1b, and the constitutive transcript is the major isoform. The subcellular distribution of rpgrip1b is distinct from its larger isoform, rpgrip1. In the photoreceptor inner segments and cells expressing enhanced green fluorescent protein (EGFP)-rpgrip1b, rpgrip1b is dispersed as punctate foci throughout the perikarya, where it colocalizes with a subpopulation of lysosomes.

Conclusions: These data support the RPGR-independent function of the isotype- and species-specific rpgrip1b in lysosome-related processes. The results further strengthen the model of the selective participation of distinct RPGRIP1 isoforms in different subcellular processes and molecular pathogenesis of RPGRIP1-allied diseases.

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Figures

Figure 1
Figure 1
Identification of a novel and abundant rpgrip1 splice variant in the murine retina. (A) Schematic diagram of mRNA splice variants encompassing exons 12 and 14 of rpgrip1 identified to date in the human (H) and the murine (M) retinas. Only a single murine rpgrip1 isoform has been identified and molecularly defined. Primers used in this study are shown as arrows below the exons. Primers P12, P14a/c, and P13c are complementary to exons 12, 14a/c, and intron 13 (398 nucleotides downstream of the exon13–intron 13 boundary). (B) RT-PCR of retinal RNA by singleplex (lanes 1 and 2) and multiplex PCR (lane 3) with primers complementary to exon 12 (P12), exon 14 (P14a/c), and/or intron 13 (P13c). The combination of P12 with P13c and P12 with P14a/c amplified amplicons of 579 and 462 bp, respectively. The former was much more abundant than the latter in the retina. (C) Sequence analysis of the amplicons indicated the 462-bp product comprised the constitutive exons 12, 13, and 14 (M12–14), whereas the 579-bp product encompassed exons 12 and an extended exon 13 (exon 13c), which contained the constitutive exon 13 and 398 bp of intron 13 (M12–13c). This splice variant of rpgrip1 produced from the transcription of intron 13 leads to the premature termination of rpgrip1 and contains three unique residues (GKS) encoded by the extended exon 13c (dashed line). (D) Amplification of rpgrip1 isoforms comprising exons 12–14 and 12–13c from a normalized mouse tissue cDNA panel. In contrast to the retina, the truncated rpgrip1b isoform was detected in low abundance only in 7-day-old embryos, the testis, and the lung among all tissues tested. Conversely, the transcript containing exons 12–14 was very abundant in the lung and testis. (E) Multiplex PCR of normalized lung and testis cDNA confirmed the results obtained with the singleplex reactions in (D). Shaded blocks in (A) and (C) represent alternative extended exons previously reported.,
Figure 2
Figure 2
Identification of a novel RPGRIP1 splice variant in the human. (A) Schematic diagram of splice variants of transcripts encompassing exons 12 and 14 of RPGRIP1. The H12–14 isoform has been reported previously. H12–13d is a new isoform of RPGRIP1 identified and reported in this work. (B) RT-PCR of retinal mRNA by singleplex (lane 2) and multiplex PCR (lane 3) with primers (shown in A) complementary to exon 12 (P12), exon 14 (P14b), and the boundary sequence between the alternatively spliced exon 13 and the constitutive exon 14 (P13d14b). Sequence analysis of the amplicons indicated that the 256- and 209-bp products comprised the constitutive exons 12, 13, and 14 (H12–14), whereas the 223- and 176-bp products encompassed exons 12 and an exon 13 (exon 13d) truncated by 33 nucleotides (H12–13d). The expression of the RPGRIP1 transcript isoform containing the canonical exons 12–14 is much higher than that observed for the other RPGRIP1 splice variant in the retina. (C) Amplification of RPGRIP1 isoforms comprising exons 12–14 and exons 12–13d from a normalized human tissue cDNA panel. All isoforms are expressed at low levels in the tissues tested. With the exception of liver, the RPGRIP1 isoform comprising exons 12–14 is not expressed or it is transcribed at much lower levels than the other RPGRIP1 isoform.
Figure 3
Figure 3
Immunoblot analysis of rpgrip1b in murine tissues. Antibodies against the seven C-terminal residues of rpgrip1b (A, B) and the coiled-coil domain of RPGRIP1/rpgrip1b (C) detected a major protein with the predicated and apparent molecular mass of 63 kDa (A) and with the expression restricted to the murine retina (B). The 63-kDa protein was not immunoreactive to the rpgrip1b Ab preadsorbed with the cognate peptide epitope and an antibody (Ab38) against the C-terminal RID domain of RPGRIP1 (data not shown). The Ab22 detected also a testis-restricted and very abundant rpgrip1 isoform (rpgrip1T110) with an apparent molecular mass of 110 kDa (D), which was also immunoreactive to an antibody (Ab38) against the C-terminal RID domain of RPGRIP1 (E).
Figure 4
Figure 4
rpgrip1 and rpgrip1b have distinct subcellular localization in the murine retina. Radial cryosections of murine retinas immunostained with two antibodies, MCW 3 (A) and MCW 4 (D), against the C2 domain of rpgrip1 and with an antibody against seven C-terminal residues of rpgrip1b (B, E). (A–C) and (D–F) are images captured at different focal planes, with the former optimally focused on the retinal cell bodies (outer [ONL] and inner [INL] nuclear layers ONL and INL) and the latter on the connecting cilium region (arrowhead) of photoreceptor neurons. (C) and (F) are merged images of (A) and (B) and of (D) and (E), respectively. Rpgrip1b was localized throughout different retinal neurons and layers (B, E) with particular preponderance in the cell bodies (ONL and INL) of these (B) and in the inner segment compartment (RIS) of rod photoreceptor neurons (E). In contrast, rpgrip1 was strongly localized to the amacrine neurons and branching processes thereof, postsynaptic sublayer of the outer plexiform layer (OPL) (A). In addition, rpgrip1 was also localized to the connecting cilium (arrowhead) of photoreceptors, external limiting membrane (arrow), and the inner segment compartment of rod photoreceptors (D). rpgrip1 and rpgrip1b had a distinct subcellular distribution pattern, as they did not colocalize in the retinal neurons (C, F). Preabsorbed anti-rpgrip1b antibody with the cognate peptide epitope completely blocked the immunostaining of the retina (E, inset). ROS, rod outer segments; OPL, outer plexiform layer; IPL, inner plexiform layer; GC, ganglion cell layer. Scale bar, 50 μm.
Figure 5
Figure 5
The EGFP-rpgrip1b fusion protein partially colocalized with a subpopulation of lysosomes in COS7 cells. Immunolabeling of EGFP-rpgrip1 fusion protein with Ab 22 against the counterpart coiled-coil domain of rpgrip1 labeled fine and aggregate foci (A) throughout the cytoplasm, which colocalized perfectly and extensively (C) with the EGFP signal of transfected cells (B). Nontransfected COS7 cells were immunonegative for rpgrip1 (not shown). The EGFP signal from the rpgrip1 fusion protein partially colocalized and interfaced closely with the lysosome-associated membrane protein Lamp1 (D–F), but not with another lysosomal marker, Lamp-2 (not shown). Screening of the double-labeled foci in transfected cells showed that the EGFP signal rarely overlapped completely with the Lamp-1-immunoreactive structures. Instead, the overlap of the signals was partial (F, inset 2). In addition, multiple and discrete EGFP signals (82% of double-labeled signals) often encircled and interfaced with a single Lamp-1-immunopositive signal (F, insets 1, 3). The EGFP-rpgrip1b fusion protein failed to colocalized with other subcellular and vesicular markers tested such as COP1 (G), EEA-1 (H), and CI-MPR (I). Scale bars: (A–I), 10 μm; (F, insets), 1 μm.
Figure 6
Figure 6
Lamp1 and rpgrip1b partially colocalized to the inner segment compartment of photoreceptor neurons. Radial cryosections of murine retinas immunostained with Lamp1 (A) and with an antibody against seven C-terminal residues of rpgrip1b (B). (C) is a merged image of (A) and (B). Lamp1 strongly localized to the inner segments (RIS) of rod photoreceptor neurons and throughout other subcellular compartments of other retinal neurons. Partial colocalization of Lamp1 with rpgrip1b was predominantly observed throughout the inner segment compartment of rod photoreceptor neurons. ROS, rod outer segments; RIS, rod inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC, ganglion cell layer. Scale bar, 50 μm.
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