A majority of mammalian genes produce multiple RNA and protein isoforms, generating extensive diversity of cellular functions (Kapranov et al., 2007
). Complex splicing patterns results in a large degree of heterogeneity of RPGR expression. Therefore, it is imperative to dissect the different protein isoforms of RPGR in the retina. We show that RPGR1-19
are detected as multiple immunoreactive bands in mammalian retina and localize to distinct subcellular compartments, likely contributing to the reported disparate localization of RPGR. Notably, these isotypes associate with different sets of proteins involved in microtubule transport in the retina.
Different isotypes generated by the RPGR1-19
splice variants can occur due to post-translational modifications (such as phosphorylation, prenylation, or proteolytic processing) or additional splicing, as demonstrated with exon ORF15 (Hong and Li, 2002
; Yan et al., 1998
). Proteolytic processing has been demonstrated for RPGRIP1 and the different products differentially localize to subcellular compartments (Lu and Ferreira, 2005
; Lu et al., 2005
). Proteolysis of ciliary proteins, such as polycystin-1 and Gli transcription factors serves as a mechanism of transducing extracellular signals to cell interior (Haycraft et al., 2005
; Low et al., 2006
; Singla and Reiter, 2006
). We previously demonstrated that nuclear proteins SMC1 and SMC3 localize to the photoreceptor connecting cilium and interact with RPGR-RLD (Khanna et al., 2005
). Given that RPGR isoforms can be detected in the nuclear fractions, we hypothesize that RPGR undergoes proteolytic processing or other post-translational modifications, resulting in association with SMC1/3 complex and transport to the nucleus as a macromolecular complex.
As demonstrated earlier for RPGRIP1 (Lu and Ferreira, 2005
), expression of multiple RPGR isoforms in the retina and their differential distribution merits the consideration of isoform ratios. It is plausible that some RPGR isoforms are enriched in specific species and cell-types, resulting in apparent differences in localization and functional compensation. Given that over-expression of a truncated RPGRORF15
isoform can result in a dominant gain-of-function effect (Hong et al., 2004
) and that a splice-site mutation upstream exon 9a of RPGR, which is associated with RP, leads to high levels of exon 9a-containing transcripts (Neidhardt et al., 2007
), abnormal enrichment of some isoforms over others may drastically change the RPGR isoform ratio and result in retinopathy.
Microsomes contain fragmented endoplasmic reticulum and are enriched in cytoskeletal proteins, metabolic enzymes, and transmembrane moieties (Han et al., 2001
). We had previously demonstrated that RPGR1-19
is prenylated at the carboxyl-terminus, a modification that assists in membrane association (Yan et al., 1998
). Although the RPGRORF15
isoform(s) do not contain the isoprenylation site (CTIL) encoded by exon 19 (Yan et al., 1998
may interact with other proteins in order to localize to microsomes.
The association of RPGR with midbody and centrosomes indicates its involvement in regulating cell division. Midbody marks the intercellular bridge during the formation of cleavage furrow at telophase (Tsvetkov et al., 2003
). It is possible that RPGR is involved in the assembly of proteins involved in the formation of the furrow by regulating microtubule-based membrane trafficking, a phenomenon critical for normal cytokinesis (Tsvetkov et al., 2003
). Support of this hypothesis comes from a previous report demonstrating the localization of another cilia-centrosomal disease protein BBS6 at midbody (Kim et al., 2005
). The precise role of RPGR in the midbody and centrosomes during cell division warrants additional studies.
In postmitotic cells, such as photoreceptors, the mother centriole of the centrosomes migrates to the apical membrane of cells and nucleates the assembly of cilia (Badano et al., 2005
). A predominant ciliary localization of both RPGRORF15
isoform may be due to the ability of the RCC1-like domain of RPGR to localize to the primary cilia. The photoreceptor connecting cilium serves as a conduit for bidirectional inter-segmental transport of protein complexes along the microtubule network (Besharse et al., 2003
; Young and Droz, 1968
). The cargo vesicles undergo polarized post-Golgi trafficking and dock at the basal bodies to be transported along the connecting cilium. This vectorial transport depends upon the activity of small GTPases, including Rab8 (Deretic et al., 1995
; Moritz et al., 2001
). Localization of RPGR1-19
isoform to the Golgi (Yan et al., 1998
) and other compartments in the inner segment (present study) may indicate a possible involvement of RPGR in post-Golgi sorting of cargo-containing vesicles for delivery to the base of the cilium. We previously showed that the carboxyl-terminal amino acids CTIL are involved in the localization of RPGR1-19
to Golgi compartments in transfected cells (Yan et al., 1998
). Given the involvement of Golgi in processing membrane proteins destined to the cilium (Cai et al., 1999
) and the localization of IFT20 to Golgi (Follit et al., 2006
), we postulate that RPGR1-19
may be involved in sorting of membrane versus soluble proteins for transport towards the connecting cilium. Owing to a lack of disease-associated mutations in exons 16-19 of RPGR1-19
isoform, this domain is either dispensable for RPGR function or mutations in this domain may lead to a lethal phenotype.
Protein trafficking via the connecting cilium is important for outer segment disc morphogenesis and renewal (Besharse et al., 2003
; Young and Droz, 1968
). Though basic components associated with the transport have been discovered (Rosenbaum, 2002
; Rosenbaum et al., 1999
), the mechanisms of cargo sorting and assembly of protein complexes, and their regulation by signaling pathways have not been elucidated. Unlike RPGRORF15
isoforms did not exhibit an association with ciliary proteins NPHP5 and IFT88 in the retina. We and others have shown that RPGR isoforms localize to the proximal region of photoreceptor connecting cilium, called the transition zone, and to basal bodies, which may act as a selection barrier for the proteins being transported distally to the axoneme and outer segments (Liu et al., 2007
; Singla and Reiter, 2006
). Moreover, RPGRORF15
do not associate with RP1, which localizes to the distal axoneme of photoreceptors (Liu et al., 2004
). Taken together, we predict that different RPGR isoforms facilitate the assembly, selection, and transport of multiprotein cargo complexes by interacting with distinct ciliary - basal body - centrosome (CBC) proteins, and that RPGR function is necessary for maintaining efficient inter-segmental transport. Further studies will be necessary to understand the composition of the CBC macromolecular complexes in the retina and the connections made by distinct RPGR isoforms within these transport particles.
We propose that diversity of RPGR isoforms is a critical determinant of its function. However, the potential of functional redundancy between RPGR1-19 and RPGRORF15 isoforms in the retina requires additional investigations aiming at delineation of the role of individual RPGR isoforms. It will also be necessary to differentiate between hypomorphic, loss-, and gain-of-function RPGR alleles to delineate the pathogenesis of photoreceptor degeneration.