Centrosomal RNA correlates with intron-poor nuclear genes in Spisula oocytes

Abstract

The evolutionary origin of centriole/kinetosomes, centrosomes, and other microtubule organizing centers (MTOCs), whether by direct filiation or symbiogenesis, has been controversial for >50 years. Centrioles, like mitochondria and chloroplasts, duplicate independently of the nucleus and constitute a heritable system independent of chromosomal DNA. Nucleic acids endogenous to the MTOC would support evolutionary origin by symbiogenesis. To date, most reports of centrosome-associated nucleic acids have used generalized reagents such as RNases and nucleic acid dyes. Here, from a library of RNAs extracted from isolated surf clam (Spisula solidissima) centrosomes, we describe a group of centrosome-associated transcripts representing a structurally unique intron-poor collection of nuclear genes skewed toward nucleic acid metabolism. Thus, we resolve the debate over the existence of centrosome-associated RNA (cnRNA). A subset of cnRNAs contain functional domains that are highly conserved across distant taxa, such as nucleotide polymerase motifs. In situ localization of cnRNA65, a molecule with an RNA polymerase domain, showed it is present in the intact oocyte nucleus (germinal vesicle). Its expression, therefore, precedes the appearance of gamma-tubulin-containing centrosomes. At this stage, the in situ signal resembles the nucleolinus, a poorly understood organelle proposed to play a role in spindle formation. After oocyte activation and germinal vesicle breakdown, cnRNA65 persists as a cytoplasmic patch within which gamma-tubulin-stained centrosomes can be seen. These observations provoke the question of whether cnRNAs and the nucleolinus serve as cytological progenitors of the centrosome and may support a symbiogenetic model for its evolution.

Fig. 1.

Differential expression of cnRNAs in centrosomes vs. whole oocyte cytoplasm. Equal quantities of RNA extracted from either whole oocyte cytoplasm or isolated centrosomes were reverse transcribed and used as template in PCR with primers targeting known cytoplasmic RNAs or putative cnRNAs. Conditions such as number of cycles, template input, and product size were determined empirically in preliminary experiments to yield product in the linear range of amplification. The examples shown here are outcomes from several separate experiments and were chosen to represent the full range of results observed. Full results, which can be cross-referenced to these images, are summarized in Table S2. Gel lanes in the upper half of the figure show PCR products obtained from cytoplasmic template for both controls (PABP, RR, 18S rRNA) and the cnRNAs (clones indicated across top). Corresponding lanes (Lower) show PCR products obtained from centrosome-derived template. A 100-bp DNA reference ladder is shown in the lanes marked Std of both Upper and Lower; 300- to 500-bp bands are marked on the left. Note that these gel images are taken from separate experiments, so a direct comparison horizontally across lanes is not a precise representation of product size.

Fig. 2.

Structural analysis of cnRNAs 15 and 194. Sense and antisense strands are defined by the orientation of the 5′ caps and 3′ polyadenylation sites. Blue bars represent the 692- and 671-nt sequences originally cloned from isolated centrosomes; arrows indicate 5′ to 3′ orientation. cnRNA15 includes a 314-nt putative protein coding domain (red) with 85% identity at the amino acid level to a newly described Spisula zinc finger protein (ZFP; GenBank accession no. AB231865). The homology domain in cnRNA15 does not correspond to the C2H2 zinc-binding motif region. In addition, a 1,128-nt domain is present in the antisense strand that exhibits 40% identity (at the amino acid level) to S. purpuratus endonuclease/RVT (GenBank accession no. XP792610). An amino acid-binding domain similar to that found in glutamate receptors and other amino acid-binding proteins from a variety of species is present in the 5′ end of cnRNA194, overlapping with a cathepsin-like cysteine protease (CysP) protease domain (green) [GenBank accession nos. EU069824 (cnRNA15) and EU069825 (cnRNA194).]

Fig. 3.

In situ localization of cnRNAs. (A) Differential interference contract microscopy (DIC) image of an unactivated oocyte labeled for cnRNA65. A dense circular patch within the GV is seen, similar in size and position to the nucleolinus. Note that both the nucleolinus and nucleolus are morphologically indistinct after cells were subjected to the hybridization regimen. A DIC image of a different unfixed oocyte is therefore shown in E for comparison with A. An arrow points to the nucleolus, and the nucleolinus is indicated by an arrowhead. B shows the distribution of cnRNA65 at 7 min postactivation. A distinct patch, although slightly more diffuse than seen in GV oocytes, is visible in the cytoplasm. Centrosomes (labeled with anti-γ-tubulin antibody in C) appear within or “attached to” the cnRNA hybridization patch. (D) Overlay of B and C. F (DIC) and G (immunofluorescence) show cnRNA65 and γ-tubulin, respectively, at 24 min postactivation (overlay in H). A series is shown for cnRNA15 in I–L: I, unactivated oocyte, arrowheads highlight ring-like hybridization patterns; J and K, cnRNA15 and γ-tubulin, respectively, at 7 min postactivation; L, overlay of J and K. (M) Hybridization control using cnRNA239 sense probe. (N) cnRNA239 (using antisense probe) at 7 min postactivation; O, γ-tubulin staining in the same cell; P, overlay. (Scale bar in P: 20 μm.]

Fig. 4.

Screen of genomic DNA for cnRNA sequences. (A Lower) Results of a PCR screen for cnRNAs 273, 278, 299, and 65 in genomic DNA extracted from oocytes (Oo), sperm (Sp), and isolated GV. (Upper) Isolated GVs from which genomic DNA was extracted are shown in the DIC micrograph. In addition to the four examples shown, DNA corresponding to 22 other cnRNAs was demonstrated to be present in all three genomic preparations. However, introns were not detected in any of the 26 cases examined. These results are diagrammed for 12 cnRNAs with amplification products of 300 bp or more (average = 460 bp) in B. The results using 12 control genes with similar PCR product sizes in cDNA (average = 482 bp) are illustrated in C. Multiple introns are revealed with canonical eukaryotic splice sequences (“Spisula consensus” in E). In subsequent analysis, cnRNAs 11 and 15 were found to be intronless >2.6-kb regions (D). Two small sequences (359 and 562 bp) were discovered within a 3.2-kb region of cnRNA194 genomic DNA (GenBank accession no. EU078900) that were not present in cDNA. (E) Sequence analysis indicates these are bona fide introns with conserved splice sites, including the invariant 5′-GU and 3′ AG residues (R = A or G; Y = C or U; W = A or T; N = any nucleotide). Results for PABP are redrawn to scale in D to highlight the relative paucity of intronic sequence in the cnRNAs vs. control Spisula sequences.