Stress-induced nuclear bodies are sites of accumulation of pre-mRNA processing factors

Abstract

Heterogeneous nuclear ribonucleoprotein (hnRNP) HAP (hnRNP A1 interacting protein) is a multifunctional protein with roles in RNA metabolism, transcription, and nuclear structure. After stress treatments, HAP is recruited to a small number of nuclear bodies, usually adjacent to the nucleoli, which consist of clusters of perichromatin granules and are depots of transcripts synthesized before stress. In this article we show that HAP bodies are sites of accumulation for a subset of RNA processing factors and are related to Sam68 nuclear bodies (SNBs) detectable in unstressed cells. Indeed, HAP and Sam68 are both present in SNBs and in HAP bodies, that we rename "stress-induced SNBs." The determinants required for the redistribution of HAP lie between residue 580 and 788. Different portions of this region direct the recruitment of the green fluorescent protein to stress-induced SNBs, suggesting an interaction of HAP with different components of the bodies. With the use of the 580-725 region as bait in a two-hybrid screening, we have selected SRp30c and 9G8, two members of the SR family of splicing factors. Splicing factors are differentially affected by heat shock: SRp30c and SF2/ASF are efficiently recruited to stress-induced SNBs, whereas the distribution of SC35 is not perturbed. We propose that the differential sequestration of splicing factors could affect processing of specific transcripts. Accordingly, the formation of stress-induced SNBs is accompanied by a change in the splicing pattern of the adenovirus E1A transcripts.

Figure 1

HAP and Sam68 colocalize in nuclear bodies both in unstressed and in heat-shocked cells. HeLa cells, either unstressed (37°C) or heat-shocked 1 h at 42°C and allowed to recover 1 h at 37°C (42°C), were costained with the anti-HAP polyclonal antibody and with the 7-1 mAb to Sam68. The distribution of HAP was revealed with an FITC-conjugated goat antirabbit antibody, whereas Sam68 was revealed with a rhodamine-conjugated goat antimouse antibody. Antigen–antibody complexes were visualized by confocal laser scanning microscopy. Colocalization of the two antigens is revealed by yellow in the merged images.

Figure 2

Identification of the region of HAP able to direct the GFP reporter protein to stress-induced SNBs. A schematic representation of the HAP structure, as detailed in the text, is shown in the top part of the figure. Regions expressed in transfected HeLa cells as GFP fusions are also indicated. Numbers refer to the first and to the last residue of each region. The ability of the different GFP fusions to associate with stress-induced SNBs was scored as + (targeting proficient) or − (targeting deficient) on the basis of the results shown in the figure. The distribution of the different GFP fusions in either unstressed (a) or heat-shocked (b) HeLa cells was determined by visualizing the autofluorescent signal of the GFP protein in confocal laser microscopy. Heat-shocked cells were also stained with the anti-HAP polyclonal antibody and the distribution of the antigen–antibody complex was revealed by indirect immunofluorescence with a rhodamine-conjugated sheep antirabbit antibody (c). Images of the same cells were merged (m) to reveal colocalization of the endogenous and of the transfected protein, resulting in yellow. The distribution of the GFP reporter protein, fused to the NLS of the simian virus 40 large T-Ag, in unstressed and heat-shocked cells is also shown. (e and f) Distribution of the endogenous HAP protein in nontransfected unstressed and heat-shocked cells, respectively.

Figure 3

The 580–788 region is necessary for the stress-induced redistribution of HAP. HeLa cells were transfected with plasmids that direct the expression either of the wild-type HAP or of a deletion mutant lacking the 580–788 region (HAP-Δ[580–788]), both fused to the HA epitope. Cells were cotransfected with a plasmid that directs the expression of GFP-[580–788], a marker of stress-induced SNBs. Transfected cells were heat shocked 1 h at 42°C and then allowed to recover 1 h at 37°C. Cells were then fixed with formaldehyde and stained with the 12CA5 mAb against the HA-epitope. The distribution of the GFP fusion was determined by visualizing the autofluorescent signal of GFP, whereas the HA-tagged protein was revealed with a rhodamine-conjugated goat antimouse IgG antibody. Confocal laser images of the same cells are shown.

Figure 4

Mapping the targeting signal within the 580–788 region by means of N-terminal or C-terminal deletion mutants. A schematic representation of the 580–788 region is shown in the top part of the figure. The gray oval indicates the coiled-coil domain. Regions expressed in transfected HeLa cells as GFP fusions are also indicated. Numbers refer to the first and to the last residue of each considered region. The ability of the different GFP fusions to associate with stress-induced SNBs was scored as + (targeting proficient) or − (targeting deficient) on the basis of the results shown in the bottom part of the figure. The distribution of the different GFP fusions in transiently transfected HeLa cells, either unstressed (a) or heat-shocked (b), was determined by visualizing the autofluorescent signal of GFP by confocal laser microscopy. Heat-shocked cells were also stained with the anti-HAP polyclonal antibody and the protein localization was revealed by indirect immunofluorescence with a rhodamine-conjugated goat antirabbit antibody (c). Images of the same cells were merged (m) to reveal colocalization of the endogenous and of the transfected protein, resulting in yellow.

Figure 5

Identification of the minimal sequence able to direct the recruitment to stress-induced SNBs. A schematic representation of the 580–788 region is shown in the top part of the figure. The gray oval indicates the coiled-coil domain. Regions expressed in transfected HeLa cells as GFP fusions are also indicated. Numbers refer to the first and to the last residue of each considered region. The ability of the different GFP fusions to associate with stress-induced SNBs was scored as + (targeting proficient) or − (targeting deficient) on the basis of the results shown in the bottom part of the figure. The distribution of the different GFP fusions in transiently transfected HeLa cells, either unstressed (a) or heat-shocked (b), was determined by visualizing the autofluorescent signal of GFP in confocal laser microscopy. Heat-shocked cells were also stained with the anti-HAP polyclonal antibody and the protein localization was revealed by indirect immunofluorescence with a rhodamine-conjugated goat antirabbit antibody (c). Images of the same cells were merged (m) to reveal colocalization of the endogenous and of the transfected protein, resulting in yellow.

Figure 6

Splicing factors SRp30c, 9G8, and SF2/ASF are recruited to stress-induced SNBs. HeLa cells were transfected with a fusion between GFP and the entire ORF of SRp30c, 9G8, or SF2/ASF. The distribution of the different GFP fusions in heat-shocked HeLa cells was determined by visualizing the autofluorescent signal of GFP in confocal laser microscopy. The same cells were stained with the anti-HAP polyclonal antibody and the protein distribution was revealed with a rhodamine-conjugated goat antirabbit antibody. As a control, nontransfected cells were heat-shocked and costained with the anti-SC35 mAb and the anti-HAP polyclonal antibody. Protein distribution was revealed with an FITC-conjugated goat antirabbit antibody and a rhodamine-conjugated goat antimouse antibody. Colocalization resulted in the appearance of yellow in the merged images.

Figure 7

Heat shock and cadmium sulfate change the alternative splicing pattern of the adenovirus E1A minigene. (A) Schematic representation of the alternative spliced isoforms of E1A pre-mRNA. The major isoforms 9S, 12S, and 13S are generated by alternative selection of the 5′ splice sites. The minor isoforms 10S and 11S involve the usage of additional internal 3′ splice sites. Sizes, in base pairs, of the corresponding RT-PCR products obtained with the use of the E1A-569 and E1A-1315 primers are shown. (B) In vivo alternative splicing of E1A pre-mRNA is affected by heat shock and by cadmium sulfate. The pCMVE1A plasmid containing the E1A minigene was transfected in HeLa cells. Twenty-four hours after transfection total RNAs were extracted from unstressed cells (37°C) or from cells heat-shocked for 1 h at 42°C. RNAs were also prepared from cells allowed to recover 1, 2, 4, or 6 h at 37°C after heat shock. RNAs were also extracted from cells treated for 3 or 5 h with 30 μM of cadmium sulfate, another induced of HAP bodies. RNA were analyzed by RT-PCR with E1A-569 and E1A-1315 primers. RT-PCR products were resolved on 5% polyacrylamide gel and detected by autoradiography. (C) ImageQuant PhosphorImager quantification of the major E1A isoforms: white bars, light gray bars, and dark gray bars, represent 13S, 12S, and 9S molecules, respectively. Percentages of the three isoforms are the average of at least three independent experiments. Error bars are shown.