Cilia and flagella are widespread cell organelles that have been highly conserved throughout evolution and play important roles in motility, sensory perception, and the life cycles of eukaryotes ranging from protists to humans. Despite the ubiquity and importance of these organelles, their composition is not well known. Here we use mass spectrometry to identify proteins in purified flagella from the green alga Chlamydomonas reinhardtii. 360 proteins were identified with high confidence, and 292 more with moderate confidence. 97 out of 101 previously known flagellar proteins were found, indicating that this is a very complete dataset. The flagellar proteome is rich in motor and signal transduction components, and contains numerous proteins with homologues associated with diseases such as cystic kidney disease, male sterility, and hydrocephalus in humans and model vertebrates. The flagellum also contains many proteins that are conserved in humans but have not been previously characterized in any organism. The results indicate that flagella are far more complex than previously estimated.

The radial spoke is a ubiquitous component of \u279+2\u27 cilia and flagella, and plays an essential role in the control of dynein arm activity by relaying signals from the central pair of microtubules to the arms. The Chlamydomonas reinhardtii radial spoke contains at least 23 proteins, only 8 of which have been characterized at the molecular level. Here, we use mass spectrometry to identify 10 additional radial spoke proteins. Many of the newly identified proteins in the spoke stalk are predicted to contain domains associated with signal transduction, including Ca2+-, AKAP- and nucleotide-binding domains. This suggests that the spoke stalk is both a scaffold for signaling molecules and itself a transducer of signals. Moreover, in addition to the recently described HSP40 family member, a second spoke stalk protein is predicted to be a molecular chaperone, implying that there is a sophisticated mechanism for the assembly of this large complex. Among the 18 spoke proteins identified to date, at least 12 have apparent homologs in humans, indicating that the radial spoke has been conserved throughout evolution. The human genes encoding these proteins are candidates for causing primary ciliary dyskinesia, a severe inherited disease involving missing or defective axonemal structures, including the radial spokes

With rare exceptions, mRNAs whose synthesis originates within nuclei contain a 3Ј poly(A) tail. Poly(A) tracts are not encoded within genes but are added to nascent pre-mRNAs in a processing reaction that involves site-specific cleavage and subsequent polyadenylation (11,16,40,42). In Saccharomyces cerevisiae, newly synthesized poly(A) tails of different transcripts are relatively homogeneous, with their final lengths determined by the combined actions of poly(A) polymerase holoenzyme (Pap1p and Fip1p), poly(A)-binding protein (Pab1p), poly(A) nuclease (PAN), and the Pab1p-associated factor, Pbp1p (10,24,43).Poly(A) tracts are generally bound by Pab1p, a highly conserved protein with four RNA recognition motifs (RRMs) connected to a carboxy-terminal helical domain via a prolineand methionine-rich segment (25,31). Association of Pab1p with poly(A) requires a minimal binding site of 12 adenosines, and multiple molecules can bind via RRMs 1 and 2 to the same poly(A) tract, spaced approximately 25 nucleotides apart (1, 2, 31, 33). In yeast, the relatively abundant 70-kDa poly(A)-binding protein is encoded by the PAB1 gene. Mutations in PAB1 cause a significant increase in the average steady-state poly(A) tail length of total cellular mRNA (32), and these effects have been attributed to two apparently nuclear functions of Pab1p: the regulation of a switch between processive and distributive activities in poly(A) polymerase (43) and the stimulation of PAN activity (7, 9, 21).Yeast poly(A) tails are initially synthesized to default lengths of 70 to 90 A's and then trimmed to mRNA-specific lengths by PAN. Analyses of three different mRNAs indicate that such trimmed tails have lengths ranging from 55 to 71 A's (8). PAN, a Pab1p-dependent 3Ј to 5Ј poly(A) exoribonuclease, requires magnesium, releases AMP as a product, and is regulated by cis-acting mRNA sequences (21). Purified PAN contains two proteins which are essential for nuclease activity: Pan2p is a 127-kDa protein with homology to the RNase T family of 3Ј35Ј exoribonucleases, while Pan3p is a 76-kDa protein which apparently acts as a positive activator of PAN activity (8). Deletion of PAN2 and/or PAN3 eliminates poly(A) nuclease activity but does not hinder cell growth. Physical interaction between Pan2p and Pan3p has been inferred from coimmunoprecipitation and two-hybrid analyses of the full-length proteins (9).Pbp1p (Pab1p-binding protein 1) specifically interacts with a 74-amino-acid segment encompassing the proline-and methionine-rich domain of the Pab1p C terminus. PBP1 is not essential for viability, but its disruption can suppress the lethality associated with a PAB1 deletion (23). In the absence of Pbp1p, 3Ј termini of pre-mRNAs are properly cleaved but receive poly(A) tails that are, on average, 15 to 30 nucleotides shorter than normal (23,25). In vitro polyadenylation reactions using extracts from wild-type and pbp1⌬ strains demonstrated that the mutant extracts initially produced full-length tails equivalent to their wild-type counterparts but subseque...

BACKGROUND: Primary ciliary dyskinesia (PCD) is a severe inherited disorder characterised by chronic respiratory disease, male infertility, and, in approximately 50% of affected individuals, a left-right asymmetry defect called situs inversus. PCD is caused by defects in substructures of the ciliary and flagellar axoneme, most commonly loss of the outer dynein arms. Although PCD is believed to involve mutations in many genes, only three have been identified. METHODS: To facilitate discovery of new PCD genes, we have used database searching and analysis to systematically identify the human homologues of proteins associated with the Chlamydomonas reinhardtii outer dynein arm, the best characterised outer arm of any species. RESULTS: We find that 12 out of 14 known Chlamydomonas outer arm subunits have one or more likely orthologues in humans. The results predict a total of 24 human genes likely to encode outer dynein arm subunits and associated proteins possibly necessary for outer arm assembly, plus 12 additional closely related human genes likely to encode inner dynein arm subunits. CONCLUSION: These genes, which have been located on the human chromosomes for easy comparison with known or suspected PCD loci, are excellent candidates for screening for disease-causing mutations in PCD patients with outer and/or inner dynein arm defects