Transcription on lampbrush chromosome loops in the absence of U2 snRNA.

Abstract

The five small nuclear RNAs (snRNAs) involved in splicing occur on the loops of amphibian lampbrush chromosomes and in hundreds to thousands of extrachromosomal granules called B snurposomes. To assess the role of these snRNAs during transcription and to explore possible relationships between the loops and B snurposomes, we injected singlestranded antisense oligodeoxynucleotides (oligos) against Ul and U2 snRNA into toad and newt oocytes. As shown before, antisense Ul and U2 oligos caused truncation of Ul and complete destruction of U2 snRNAs, respectively. However, injection of any oligo, regardless of sequence, brought on dramatic cytological changes, including shortening of the chromosomes and retraction of the lateral loops, with concomitant shutdown of polymerase II transcription, as well as disappearance of some or all of the B snurposomes. When injected oocytes were incubated for 12 h or longer in physiological saline, these changes were reversible; that is, the chromosomes lengthened, transcription (detected by 3H-UTP incorporation) resumed on newly extended lateral loops, and B snurposomes reappeared. In situ hybridization showed that loops and B snurposomes had negligible amounts of U2 snRNA after recovery from injection of the anti-U2 oligo, whereas these structures had normal levels of U2 snRNA after recovery from a control oligo. Thus, the morphological integrity of B snurposomes and lampbrush chromosome loops is not dependent on the presence of U2 snRNA. Because transcription occurs in the absence of U2 snRNA, we conclude that splicing is not required for transcription on lampbrush chromosome loops.

The Tripartite Motif of Nuclear Factor 7 Is Required for Its Association with Transcriptional Units

“…Interestingly, another component of the RNP matrix, the U1 snRNP, is normally detected in oocyte CBs by in situ hybridization at a level slightly higher than background levels obtained with control probes (42). Yet, upon truncation of its first 20 nucleotides, endogenous U1 accumulates in CBs (39). Because the splicing snRNPs traffic through CBs (27), one likely interpretation is that the exchange kinetics of U1 between CBs and the nucleoplasm are changed, resulting in an accumulation of U1 in CBs.…”

Splicing components are excluded from the transcriptionally inactive XY body in male meiotic nuclei.

“…This positional relationship between sites of transcription and sites of pre-mRNA splicing is supported by a large body of observations at the light and electron microscope level (reviewed in Gall, 1992;Spector, 1993;Xing and Lawrence, 1993;Kramer et al, 1994). In some cases, the pre-mRNAs that are found associated with splicing components in these nuclear sites are in a nascent form (Beyer and Osheim, 1988;Gall and Callan, 1989;Vazques-Nin et al, 1990;Wu et al, 1991;Tsvetkov et al, 1992;Jimenez-Garcia and Spector, 1993;Wansink et al, 1993;Xing and Lawrence, 1993;Zachar et al, 1993). In other cases, the premRNA is found at various stages of the RNA processing pathway (Fakan et al, 1984;Carter et al, 1991;Xing and Lawrence, 1993;Kramer et al, 1994).…”

Stable intronic sequence RNA (sisRNA), a new class of noncoding RNA from the oocyte nucleus of Xenopus tropicalis

“…Transcription was inhibited by incubating pieces of ovary in actinomycin D (20 mg/mL) in OR2 medium. Splicing was inhibited by injecting individual oocytes with a single-stranded deoxyoligonucleotide complementary to part of the U2 snRNA molecule (Tsvetkov et al 1992). …”

“…Earlier experiments showed that Xenopus oocytes can be depleted of U2 by injecting a specific antisense deoxyoligonucleotide . The snRNA is reduced to <1% of its original amount within 10-30 min and remains at this level for at least 48 h (Tsvetkov et al 1992). Under these conditions, splicing does not occur, and the normally abundant U2 snRNA is not detectable on the lampbrush chromosome loops by in situ hybridization.…”

“…Under these conditions, splicing does not occur, and the normally abundant U2 snRNA is not detectable on the lampbrush chromosome loops by in situ hybridization. However, transcription continues for several days (Tsvetkov et al 1992).…”