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Xenopus laevis short interspersed repeat transcripts (Xlsirts) are a family of noncoding RNAs defined by the presence of a specific repeated sequence that acts as a vegetal localization element. Previous studies have demonstrated that Xlsirts function as localization elements to localize RNA and also in anchoring mRNA at the vegetal cortex. However, the identity of the Xlsirts containing family members present at the cortex was unknown. We identified 17 new Xlsirt cDNAs from an oocyte cDNA library. In addition to being associated with noncoding sequences, the repeats were also present in cDNAs with open reading frames. Xlsirt RNAs with repeats in the correct orientation were capable of localizing to the vegetal cortex. Our observations demonstrate that a heterogeneous population of Xlsirt RNAs is present at the cortex and that this population contains both noncoding RNAs and RNAs encoding proteins that are likely to play important roles in the subsequent development of the embryo.
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12676327
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Fig. 2. RT-PCR analysis of stages III oocyte RNA. Detecting Xlsirt clones indicates that these cDNAs are representative of RNAs present in the oocyte. (A)
Diagram of Xlsirt RNA sequences with the position of PCR primers indicated by bars over and under the RNA. The position of open reading frames is indicated
by RF The repetitive region of each RNA is shaded in gray. (B) Ethidium bromide-stained gel: (III) RT-PCR of clones Xlsirt-tran, (Xlsirt-unk1, and
Xlsirt 4-8 from stages I to III oocyte RNA. (2) no RT controls for each clone. ( positive control PCR reactions of plasmid DNA containing cDNA insert
amplified with the same primer pair. Autoradiograph: (III) RT-PCR of Xlsirt-met from stages I to III oocyte RNA. (2 ) RT-PCR starting with twice the
amount of stage III RNA. (21) negative control with no reverse transcriptase. (22) negative control with no PCR.
Fig. 3. Localization of in vitro synthesized transcripts of four cDNA clones
microinjected into X. laevis oocytes. (A), sections of stage III oocytes.
Bar 125 mm. (E,F), whole stage III oocytes. Bar 250 mm. (A) Control
injection of a nonlocalizing RNA, the antisense strand of Xlsirt-non1; (B)
Xlsirt-unk1, which contains repeats in the reverse orientation and does not
show localization; (C) Xlsirt 4-8, which contains forward repeats and shows
localization; (D) Xlsirt-trans, which contains forward repeats and shows
localization; (E) Control injection of Xlsirt-met synthetic transcript lacking
repetitive region; (F) Xlsirt-met, which contains forward repeats and shows
localization.
