Splicing-independent recruitment of spliceosomal small nuclear RNPs to nascent RNA polymerase II transcripts

Abstract

In amphibian oocytes, most lateral loops of the lampbrush chromosomes correspond to active transcriptional sites for RNA polymerase II. We show that newly assembled small nuclear ribonucleoprotein (RNP [snRNP]) particles, which are formed upon cytoplasmic injection of fluorescently labeled spliceosomal small nuclear RNAs (snRNAs), target the nascent transcripts of the chromosomal loops. With this new targeting assay, we demonstrate that nonfunctional forms of U1 and U2 snRNAs still associate with the active transcriptional units. In particular, we find that their association with nascent RNP fibrils is independent of their base pairing with pre-messenger RNAs. Additionally, stem loop I of the U1 snRNA is identified as a discrete domain that is both necessary and sufficient for association with nascent transcripts. Finally, in oocytes deficient in splicing, the recruitment of U1, U4, and U5 snRNPs to transcriptional units is not affected. Collectively, these data indicate that the recruitment of snRNPs to nascent transcripts and the assembly of the spliceosome are uncoupled events.

Figure 1.

Newly assembled spliceosomal snRNPs associate rapidly with CBs and IGCs (B-snurposomes). Differential interference contrast (DIC) and corresponding fluorescent micrographs of nuclear spreads from oocytes injected with fluorescent U1 or U7 snRNAs, respectively (green). Organelles are readily distinguished by their morphology with DIC and specific probes. Here, the DNA-specific dye Syto61 was used to labeled nucleoli (red), whereas the anticoilin antibody (mAb H1) was used to label CBs (blue; arrows). Newly made fluorescent U1 snRNP is detected in both CBs and IGCs (asterisks) as early as 1 h after cytoplasmic injection of fluorescent U1 snRNA. In contrast, a newly assembled fluorescent U7 snRNP, which is not involved in splicing, accumulates exclusively within CBs. In both cases, nucleoli are negative. Bars, 5 μm.

Figure 2.

Association of newly made fluorescent spliceosomal snRNPs with active transcriptional units. In vitro transcribed snRNAs were injected into the cytoplasm of stage V oocytes, and nuclear spreads were prepared 18 h later. In all preparations, the DNA was counterstained with DAPI, which is pseudocolored here in red. The fluorescent snRNA signal is shown in green. (A and B) A phase-contrast image and its corresponding fluorescent image are presented for the U1 snRNP. The inset in B corresponds to a laser-scanning confocal image of several chromosomal loops showing association of the U1 snRNP with the nascent RNP fibrils. (C–F) Fluorescent images are shown for U7, U2, U4, and U5 snRNPs. Consistent with the distribution of the endogenous splicing snRNPs, the newly assembled U1, U2, U4, and U5 snRNPs were detected in IGCs, CBs (arrows), and on the loops of LBCs. (C) The nonspliceosomal U7 snRNP accumulated in CBs but was absent from IGCs and the chromosomal loops. Note that DAPI labels well the nucleoli and, to a lesser extent, IGCs (most likely because of their high content in RNAs), which are structures of ∼1 μm in diameter. DAPI also labels well the chromosomal axes, which correspond to transcriptionally inactive domains. Bar, 10 μm.

Figure 3.

Newly assembled snRNPs associate with RNAPII but not RNAPIII nascent transcripts. Phase-contrast and corresponding fluorescent micrographs of nuclear spreads from oocytes injected 18 h earlier with fluorescent U1 snRNA (green). (A) Oocytes were treated with actinomycin D (AMD) for 1 h before nuclear spread preparation. Phase contrast shows one of the 18 LBCs, which are devoid of lateral loops as a result of transcription inhibition. Fluorescent U1 snRNP associates with CBs (arrow) and IGCs but fails to target chromosomes. The chromosomal axis and nucleoli are counterstained with DAPI (pseudocolored in red). (B) An anti-RPC53 antibody was used to identify the ∼90 RNAPIII transcriptional sites. One such RNAPIII locus is shown here (red) to illustrate the fact that newly assembled fluorescent U1 snRNPs are not recruited there. Note that this locus is not visible by phase contrast. If many RNAPIII loci appear as rather amorphous structures like the one presented here, several others tend to display long lateral loops. One such loop is presented in the inset at the same magnification. Notice that no green signal is associated with the loop. Bars, 5 μm.

Figure 4.

Mutant U1 and U2 snRNAs that cannot engage splicing are still recruited to active transcriptional units. U1(ΔSS) and U2(ΔBPS) snRNAs (green) were injected into the cytoplasm of stage V oocytes, and nuclear spreads were prepared 18 h later. (A) Diagram shows the regions of the U1 and U2 snRNAs involved in interacting with pre-mRNA. (B) Diagram shows the regions deleted (red dashed lines) in the mutant U1(ΔSS) and U2(ΔBPS) snRNAs. Laser-scanning confocal images showing the association of U1(ΔSS) and U2(ΔBPS) snRNPs with the nascent transcripts of the chromosomal loops. Bar, 5 μm.

Figure 5.

A nonfunctional U2 snRNP targets nascent RNP fibrils. Phase contrast or DIC and corresponding fluorescent micrographs of nuclear spreads from an oocyte injected 18 h earlier with fluorescent U2(Δ29) snRNA (green). The newly assembled U2(Δ29) snRNP is recruited to CBs (arrow), IGCs, and the chromosomal loops. Note that the labeling of CBs is dramatically reduced when compared with full-length U2 snRNA (Fig. 2). Chromosomal axes and nucleoli were counterstained with DAPI (pseudocolored in red). A diagram shows the deleted region of U2 snRNAs (dashed red line). Bars, 5 μm.

Figure 6.

The first stem loop of the U1 snRNA is necessary and sufficient for its association with IGCs and nascent RNP fibrils. (A and B) Phase contrast or DIC and corresponding fluorescent micrographs of nuclear spreads from oocytes injected 18 h earlier with either mutant U1(Δ47) RNA (A) or chimeric U7/U1(I) RNA (green; B). The diagram above each panel indicates the structure of the corresponding RNAs. The deleted residues in U1(Δ47) are indicated by a dashed line. Stem loop I is colored in red. The newly assembled U1(Δ47) snRNP targets CBs (arrows) but fails to associate with chromosomal loops and IGCs. In contrast, the U7/U1(I) snRNP associates with nascent RNP fibrils and IGCs in addition to CBs. A group of CBs, IGCs, and nucleoli are presented at a higher magnification in both cases. Note that the signal resulting from the association of U7/U1(I) snRNP with CBs is very weak. DAPI (pseudocolored in red) was used here to counterstain nucleoli and chromosomal axes. Note that IGCs are weakly labeled because of their high RNA content. Bars, 10 μm.

Figure 7.

Depletion of the U2 snRNA inhibits U2B″ targeting to chromosomal loops. (A) Northern blot analysis indicates that U2 snRNA is completely depleted in U2b oligonucleotide–injected oocytes but is unaffected in control oocytes that were injected with the control oligonucleotide or just water. Each lane was loaded with the total RNA fraction of one nucleus isolated 18 h after injection. U5 snRNA was used here as a loading control. (B) Fluorescent micrographs of nuclear spreads prepared 18 h after injection with either the U2b or C oligonucleotide. The U2-specific protein U2B″ was detected using mAb 4G8 (red). In control oocytes, U2B″ is found associated with the nascent transcripts of the chromosomal loops as well as with IGCs. Nucleoli are also weakly stained. In U2b- injected oocytes, U2B″ is no longer detected on the chromosomal loops or the IGCs. Instead the granular region of nucleoli is brightly labeled. DAPI is pseudocolored in green. Bar, 5 μm.

Figure 8.

Newly formed fluorescent U2 snRNP rescues the association of U2B″ with nascent transcripts and IGCs in U2-depleted oocytes. Phase contrast and corresponding fluorescent micrographs of nuclear spreads from oocytes injected with U2b and fluorescent U2 snRNA as indicated in the diagram. U2B″ was detected using mAb 4G8 (red) and displays an extensive colocalization with fluorescent U2 snRNA (green) on both chromosomal loops and IGCs. Bars, 2 μm.

Figure 9.

Y14 is not recruited to nascent transcripts in the absence of the U2 snRNA. (A) The fate of newly expressed HA-Y14 was followed in stage V oocytes using the anti-HA antibody mAb 3F10 48 h after the injection of its corresponding transcript. A single band of ∼24 kD, which is primarily nuclear, is detected on immunoblots. On nuclear spreads, HA-Y14 (green) associates strongly with CBs (arrow) as well as with the nascent transcripts on chromosomal loops. IGCs and the dense fibrillar region of nucleoli are weakly stained. (B) Phase contrast and corresponding fluorescent micrographs of nuclear spreads from oocytes coinjected with HA-Y14 transcripts and either the C or the U2b oligonucleotide. In the rescue experiment, fluorescent U2 snRNA was injected 18 h later. All nuclear spreads were prepared 48 h after the initial injections. The distributions of HA-Y14 (green) and U2B″ (red) were defined using mAb 3F10 and mAb 4G8, respectively. In U2 snRNA–depleted oocytes (U2b injected), HA-Y14 is still found within CBs (arrow), IGCs, and nucleoli, but it is absent from chromosomal loops. In these oocytes, U2B″ accumulates within nucleoli. Remarkably, the chromosomal association of HA-Y14 is rescued by the fluorescent U2 snRNA (pseudocolored in red). Bars, 5 μm.

Figure 10.

Spliceosomal U1, U4, and U5 snRNPs target LBC loops in the absence of splicing. Fluorescent U1, U4, or U5 snRNAs were injected into the cytoplasm of stage V oocytes previously depleted of their endogenous U2 snRNA. Nuclear spreads were prepared 18 h later, and the distribution of the newly assembled snRNPs (green) was determined by fluorescence microscopy. (A) In all three cases, a signal was associated with the chromosomal loops as well as with CBs (arrows) and IGCs. Thus, targeting of the U1 snRNP and the U4/U6.U5 tri-snRNP to nascent transcripts does not require the presence of the U2 snRNP. U2B″ (red) is detected using mAb 4G8 and is found accumulated in the granular region of nucleoli, which is indicative of an efficient U2 snRNA depletion. (B) Magnified views (laser-scanning microscopy) of particularly extended chromosomal loops that illustrate the association of newly assembled snRNPs with nascent transcripts. Bars (A), 10 μm; (B) 1 μm.