Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes

Abstract

The important role that cilia and flagella play in human disease creates an urgent need to identify genes involved in ciliary assembly and function. The strong and specific induction of flagellarcoding genes during flagellar regeneration in Chlamydomonas reinhardtii suggests that transcriptional profiling of such cells would reveal new flagella-related genes. We have conducted a genome-wide analysis of RNA transcript levels during flagellar regeneration in Chlamydomonas by using maskless photolithography method-produced DNA oligonucleotide microarrays with unique probe sequences for all exons of the 19,803 predicted genes. This analysis represents previously uncharacterized wholegenome transcriptional activity profiling study in this important model organism. Analysis of strongly induced genes reveals a large set of known flagellar components and also identifies a number of important disease-related proteins as being involved with cilia and flagella, including the zebrafish polycystic kidney genes Qilin, Reptin, and Pontin, as well as the testis-expressed tubby-like protein TULP2.cilia ͉ flagella ͉ polycystic kidney disease ͉ RNA transcription C ilia are sensory and motile structures that play critical roles in animal physiology and development. Ciliary defects have been implicated in several human genetic diseases, including polycystic kidney disease (PKD), Bardet-Biedl Syndrome (BBS), and primary ciliary dyskinesia (also known as Kartagener's Syndrome) (1). The symptoms of these diseases include physiological defects such as kidney failure, defective mucus clearance, neurosensory deficits, and obesity, as well as developmental defects such as situs inversus and polydactyly. To understand the role of cilia in development and physiology, it is essential to identify the gene products necessary for ciliary assembly and function. A proteomic analysis of human cilia has been presented (2), but such analyses are limited in their ability to detect low-abundance proteins. Two recent studies used comparative genomics to identify genes found only in the genomes of organisms that contain cilia and flagella (3, 4), and the set of genes thus identified is referred to as the flagellar and basal body proteome. Those bioinformatics studies detected several disease genes related to cilia, including several genes mutated in BBS patients. The success of the comparative genomics approach in identifying BBS genes suggests that analysis of the components of cilia and flagella would be an effective way to identify additional genes involved in ciliary diseases.In this report, we take an alternative strategy for identifying ciliary components, by taking advantage of the fact that most known components of cilia and flagella are strongly induced during regeneration of flagella in Chlamydomonas reinhardtii (5). Chlamydomonas is a green alga with genetics similar to yeast, but unlike yeast, it has flagella that are virtually identical to human cilia. Most of the previously identified human ciliary disease genes have orthologs in Chlamydomo...

Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

“…Although IFT38 was not identified in the pioneering characterizations of the IFT‐B complex (Piperno & Mead, 1997; Cole et al , 1998), several lines of evidence suggest that IFT38 is a bona fide IFT‐B complex protein. IFT38 localizes to the cilium and is required for ciliogenesis in a wide range of organisms, and mutations in IFT38 cause cystic kidneys in zebrafish and result in a loss of hedgehog signaling and embryonic lethality in mice (Sun et al , 2004; Stolc et al , 2005; Absalon et al , 2008). Furthermore, IFT38 was shown to undergo IFT in C. elegans (Ou et al , 2005) and co‐precipitated along with other IFT‐B proteins from zebrafish cell lysates using TAP‐IFT54 as a bait (Omori et al , 2008).…”

LRRC50, a Conserved Ciliary Protein Implicated in Polycystic Kidney Disease

“…LRRC50 was identified as a putative ciliary protein in two independent bioinformatic studies. 11,12 Reminiscent of the localization of other PKD-associated proteins (for a comprehensive overview, see reference 10 ), we showed localization of human LRRC50-EGFP at the mitotic spindle poles and cilium in human and dog kidney cells, suggesting LRRC50 to be a ciliary component in vertebrates.…”

“…Cilia are now perceived as important cellular antennae, and mechanisms underlying ciliogenesis and cilia maintenance have recently become a major focus of research, including systematic bioinformatic screens to define a comprehensive ciliary proteome or ciliome. 4,[11][12][13][14][15][16] In search for novel genes essential for ciliary motility, we performed a forward genetic N-ethyl-N-nitrosourea (ENU) screen in zebrafish. Here, we report the isolation of a mutant with ciliary dyskinesia that develops proliferative kidney cysts.…”

IFT‐81 and IFT‐74 are required for intraflagellar transport in C. elegans

“…Many comprehensive studies using bioinformatics, genomics and proteomics have identified numerous putative ciliary proteins (Ostrowski et al 2002;AvidorReiss et al 2004;Li et al 2004;Blacque et al 2005;Efimenko et al 2005;Pazour et al 2005;Smith et al 2005;Stolc et al 2005). A recent work showed that a cGMP-dependent protein kinase (PKG) depending on IFT machinery participate in the flagellar-adhesion pathway in the alga Chlamydomonas, suggesting IFT has a direct role in cilium-based signaling (Scholey & Anderson 2006;Wang et al 2006;Yoder 2006).…”