Talhouarne GJ and Gall JG (2014), Lariat intronic RNAs in the cytoplasm of Xenopu...

XB-ART-49281

RNA 2014 Sep 01;209:1476-87. doi: 10.1261/rna.045781.114.

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Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes.

Talhouarne GJ , Gall JG .

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We previously demonstrated that the oocyte nucleus (germinal vesicle or GV) of Xenopus tropicalis contains a population of stable RNA molecules derived from the introns of most expressed genes. Here we show that similar stable intronic sequence (sis) RNAs occur in the oocyte cytoplasm. About 9000 cytoplasmic sisRNAs have been identified, all of which are resistant to the exonuclease RNase R. About half have been confirmed as lariat molecules and the rest are presumed to be lariats, whereas nuclear sisRNAs are a mixture of lariat and linear molecules. Cytoplasmic sisRNAs are more abundant on a molar basis than nuclear sisRNAs and are derived from short introns, mostly under 1 kb in length. Both nuclear and cytoplasmic sisRNAs are transmitted intact to the egg at GV breakdown and persist until at least the blastula stage of embryogenesis, when zygotic transcription begins. We compared cytoplasmic sisRNAs derived from orthologous genes of X. tropicalis and X. laevis, and found that the specific introns from which sisRNAs are derived are not conserved. The existence of sisRNAs in the cytoplasm of the oocyte, their transmission to the fertilized egg, and their persistence during early embryogenesis suggest that they might play a regulatory role in mRNA translation.

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Species referenced: Xenopus tropicalis Xenopus laevis

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	FIGURE 1. Characterization of cytoplasmic sisRNAs. (A) The oocyte nucleus or germinal vesicle (GV) can be removed from a defolliculated oocyte within seconds to provide a sample of cytoplasm uncontaminated with nuclear RNA. (B) IGV browser view of a typical gene bearing a cytoplasmic sisRNA. Cytoplasmic intronic reads (blue) occur in one intron of the eif4a1 gene (yellow exonic reads). The lower track (track height 100 reads) shows that the intronic reads do not cross the intron–exon borders. The yellow and blue arrows represent exonic and intronic primers used for RT–PCR. (C) RT–PCR results showing that cytoplasmic sisRNAs are derived from the sense strand and do not cross the intron–exon borders. (ivt) In vitro transcribed RNA, used as a control template. (D) Comparison of cytoplasmic, nuclear (GV), and whole oocyte RNAs from the rbm28 gene. Cytoplasmic and whole oocyte RNAs are essentially identical (arrow points to cytoplasmic sisRNA, blue reads). Nuclear sisRNAs (red reads in GV track) are not detectable in the whole oocyte sample. (E) Distribution of sisRNAs relative to intron length. The majority of cytoplasmic sisRNAs (blue) derive from shorter introns, whereas nuclear-specific sisRNAs (red) come from a wide range of longer introns.
	FIGURE 2. Cytoplasmic sisRNAs are resistant to RNase R. (A) Cytoplasmic RNA was treated with RNase R or water and then used for RT–PCR amplification of exonic and intronic sequences from three genes. Intronic, but not exonic, sequences were resistant to RNase R and could still be amplified. The same intronic sequences (ivt RNA), when derived from in vitro-transcribed RNA, were digested by RNase R and did not amplify. (B) Cytoplasmic RNA was digested with RNase R and subjected to deep sequencing. The upper track (control) shows that cytoplasmic sisRNA sequences (blue) are rare relative to the coding sequences (yellow) of the nasp gene. The lower track (RNase R treated) shows that the sisRNA sequences are resistant to the enzyme, whereas the mRNA is almost completely digested. (C) A relatively rare case in which an mRNA is resistant to RNase R digestion (lpcat4). Presumably, digestion began at the 3′ end of the mRNA but could not proceed past the middle of the last exon. (D) A circular molecule derived from two exons without the intervening intron in the gene lsm1. Such molecules could arise by “backsplicing,” as recently described for human fibroblasts (Jeck et al. 2013).
	FIGURE 3. Evidence that cytoplasmic sisRNAs are circles (lariats without tails). (A) Plot of average read number per nucleotide for 200 sisRNAs relative to nucleotide position within the intron. Reads abut the 5′ end of the intron, but are absent from a region of ∼30 nt next to the 3′ end. Consensus nucleotides are shown for positions near the ends of the introns. (B) Inverted reads contain sequences that cross the splicing branchpoint and can be mapped when appropriately split (Taggart et al. 2012). (C) Some inverted reads contain sequence errors or nucleotides inserted at the branchpoint (red nucleotides), presumably by the reverse transcriptase used for library production. (D) Cytoplasmic sisRNAs yield an RT–PCR product when amplified with outward-facing primers. These same primers amplify in vitro-transcribed RNA only when the branchpoint is in the interior of the molecule. See text for details of cloning and sequencing of the RT–PCR products.
	FIGURE 4. (A,B) The stability of X. tropicalis cytoplasmic RNA (1 µg) was tested by RT–PCR after incubation in X. laevis cytoplasmic or nuclear extracts. (A) Extracts of X. laevis whole oocytes, oocyte cytoplasm, and GVs were made under oil. (B) Cytoplasmic sisRNAs from X. tropicalis were degraded by a GV extract from X. laevis but not by a cytoplasmic extract (intron primer set, blue). Exonic sequences from X. tropicalis were stable for 2 h in both GV and cytoplasmic extracts (exon primer set, yellow). (C,D) Splicing activity was tested by injecting an in vitro-transcribed X. tropicalis pre-mRNA construct into the cytoplasm or GV of an X. laevis oocyte. (C) Injection of the construct was carried out under mineral oil. (D) After incubation for 2 h, an RT–PCR reaction was carried out on the cytoplasm and GV using primers from the ends of the construct. The products were run on an agarose gel and stained. Splicing occurred only in the GV, as shown by the expected smaller size of the RT–PCR product.
	FIGURE 5. During oogenesis cytoplasmic sisRNAs but not snoRNAs increase in abundance relative to mRNAs. (A) Cytoplasmic sisRNAs (blue) occur in three introns of the tnpo2 gene (top track, cytoplasm from mature oocyte). During oogenesis these sisRNAs increase in abundance relative to exonic sequences (four lower tracks, total RNA from oocytes of increasing size). (B) During oogenesis two nuclear snoRNAs (green) maintain a roughly constant ratio relative to cytoplasmic exonic sequences (yellow). The arrow points to a snoRNA whose size and abundance is similar to that of the sisRNAs detected in A. The exclusively nuclear location of the snoRNA sequences is shown by their absence from cytoplasm of mature oocytes (top track).
	FIGURE 6. Persistence of sisRNAs in the early embryo. (A) A prominent cytoplasmic sisRNA in the ckap5 gene (top track, blue) persists in the egg, 4-cell, and early blastula stages. (B) The top two tracks show nuclear sisRNAs (red) in the GV and cytoplasmic sisRNAs (blue) in the cytoplasm of mature oocytes. The third track shows that cytoplasmic sisRNAs (blue) are resistant to RNase R relative to the mRNA (yellow). Note that nuclear sisRNAs are not seen in this cytoplasmic sample, attesting to its purity. The bottom three tracks show egg, 4-cell, and early blastula RNA treated with RNase R. These samples show persistence not only of cytoplasmic sisRNAs (blue) but also of nuclear sisRNAs (red) derived from GV breakdown at the time of fertilization.
	FIGURE 7. Diagram of a Xenopus oocyte showing proposed origin of nuclear and cytoplasmic sisRNAs.

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