Spatial regulation of cytoplasmic snRNP assembly at the cellular level

Abstract

Small nuclear ribonucleoproteins (snRNPs) play a crucial role in pre-mRNA splicing in all eukaryotic cells. In contrast to the relatively broad knowledge on snRNP assembly within the nucleus, the spatial organization of the cytoplasmic stages of their maturation remains poorly understood. Nevertheless, sparse research indicates that, similar to the nuclear steps, the crucial processes of cytoplasmic snRNP assembly may also be strictly spatially regulated. In European larch microsporocytes, it was determined that the cytoplasmic assembly of snRNPs within a cell might occur in two distinct spatial manners, which depend on the rate of de novo snRNP formation in relation to the steady state of these particles within the nucleus. During periods of moderate expression of splicing elements, the cytoplasmic assembly of snRNPs occurred diffusely throughout the cytoplasm. Increased expression of both Sm proteins and U snRNA triggered the accumulation of these particles within distinct, non-membranous RNP-rich granules, which are referred to as snRNP-rich cytoplasmic bodies.

Keywords: Cajal bodies; Larix; P bodies; Sm proteins; confocal microscopy; in situ hybridization; plant cell; snRNA; splicing..

Fig. 1.

Quantitative analysis of Sm protein (A) and U4 snRNA (B) cytoplasmic and nuclear levels during diplotene in larch microsporocytes. Relative fluorescence intensity is given as arbitrary units (a.u.) per cell. Five similar cycles of the amount and distribution of the molecules are highlighted. Each cycle consisted of three to five stages, named A–E. Asterisks represent stages of cytoplasmic snRNP assembly during each cycle. Results are shown as means±SE.

Fig. 2.

Double labelling of U4 snRNA and Sm proteins during the fourth cycle of synthesis. Stages A and B are divided in two substages referred to as A₁ and A₂ for stage A and B₁ and B₂ for stage B because of the distinct snRNP patterns of localization. (A–D) Stage A₁. Nuclear U4 snRNA fluorescence was visible (A), most of which colocalized with the Sm signal (B, C). Additionally, spherical 2 µm diameter foci of Sm signal accumulation were observed in the cytoplasm (B, arrowheads), lacking U4 snRNA (C, arrowheads). (E–H) Stage A₂. The U4 snRNA signal localized to the entire nucleus, with noticeable irregular accumulations of transcripts (E) devoid of the Sm signal (F, G). In the cytoplasm, distinguishable foci of Sm accumulation remained visible (F, G, arrowheads). (I–L) Stage B₁. The U4 snRNA staining indicated a significant enrichment of transcripts at the border between the nucleus and cytoplasm (I, asterisk), lacking corresponding Sm accumulation (J, K, asterisk). Within the cytoplasm, the U4 snRNA was localized in large cytoplasmic clusters (I, arrowheads, inset I’), which colocalized with Sm proteins (J, K, arrowheads, insets J’, K’). (M–P) Stage B₂. Strong nuclear U4 snRNA and Sm staining showed a dispersed pattern of localization within the nucleoplasm, with distinct accumulation in individual CBs that formed in close proximity to the nuclear envelope (M–O). No cytoplasmic clusters of U4 snRNA fluorescence were visible (M), whereas Sm staining still exhibited numerous Sm-rich cytoplasmic granules (N, O, arrowheads). (R–U) Stage C. The nuclear signal of U4 snRNA and Sm fluorescence showed a more evenly distributed pattern within the nucleoplasm (R, S); the CBs were located throughout the whole nucleus and were frequently associated with the nucleolus (R–T). In the cytoplasm, there were Sm-containing clusters that remained visible (S, T, arrowheads), which lacked U4 snRNA (R). The corresponding DAPI images were collected using wide-field fluorescence (D, H, L, P, U). nu, Nucleolus. Bars, 10 µm.

Fig. 3.

Double labelling of U4 snRNA and Sm proteins during the fifth cycle of synthesis. (A–D) Stage A. Nuclear U4 snRNA staining was visible (A), with a noticeable portion of nucleoplasmic signals devoid of Sm accumulation (B, C). The cytoplasmic signal from Sm localization was dispersed throughout the cytoplasm, and no accumulation of Sm in distinct clusters was present (B). (E–H) Stage B. The U4 snRNA signal showed a diffused pattern of localization within the nucleoplasm (E); it was also enriched in numerous CBs, which colocalized with Sm staining (E–G). The cytoplasmic Sm localization was dispersed, with numerous discrete accumulations (F, G, arrowheads, insets F’, G’), lacking U4 snRNA (E, inset E’). (I–L) Stage C. An increased level of nuclear Sm staining was visible, which colocalized with U4 snRNA in both the nucleoplasm and the CB (I–K). In the cytoplasm, the Sm fluorescence signal indicated only a dispersed pattern (J). The corresponding DAPI images were collected using wide field fluorescence (D, H, L). Bars, 10 µm.

Fig. 4.

Colocalization of U5 snRNA and Sm proteins during late diplotene. (A–D) Stage B₁ during the fourth cycle of synthesis. Numerous cytoplasmic foci enriched in both U5 snRNA (A, arrowheads, inset A’) and Sm (B, arrowheads, inset B’) are visible (C, arrowheads, inset C’). (E–H) Stage B during the fifth cycle of synthesis. The cytoplasmic signal from Sm localization was dispersed throughout the cytoplasm with discrete accumulations (F, G, arrowheads, insets F’, G’) that lacked U5 snRNA (E, inset E’). The corresponding DAPI image was collected using wide field fluorescence (D, H). Bars, 10 µm.

Fig. 5.

Colocalization analysis of splicing elements during the cytoplasmic stages of snRNP assembly. Columns representing the percentage of U4 snRNA that colocalized with Sm proteins relative to the entire cytoplasmic pool of transcripts, and the percentage of Sm proteins that colocalized with U4 snRNA relative to the entire cytoplasmic pool of the proteins are indicated. Results are shown as means±SE. (This figure is available in colour at JXB online.)

Fig. 6.

Analysis of the cytoplasmic:nuclear ratio of U4 snRNA and Sm proteins in larch microsporocytes during the stages of cytoplasmic snRNP assembly. Asterisks represent the stages of accumulation of the investigated molecules in distinct cytoplasmic clusters.