Heterogeneous ribonucleoproteins A1 and A2/B1 (hnRNP A1 and A2/B1)

apoptotic nuclei.

To test changes due to apoptosis in intact cells, HeLa cells were stimulated with TRAIL.

Immunocytochemistry was performed with two antibodies, one raised against a peptide common to all hnRNP A/B splicing variants (KEDTEEHHLRDYFE) and the other specific for hnRNP A1 (clone 4B10, immunogen: full length native protein partially purified) to at least one specific hnRNP A isoform. An antibody specific for hnRNP A2/B1 was not available. The specimens were imaged at the confocal microscope, and the protein distribution within the cells was analysed by plotting distribution profiles specific for hnRNP A (green line) isoforms and for chromatin (red line). The general anti-hnRNP A/B antibody in untreated control cells revealed a dominant nuclear staining with a clear omission of the nucleoli. Thirty minutes after induction of apoptosis, an increase of the cytosolic hnRNP A/B signal could be detected, which was accompanied by the formation of foci in the cytosol (Fig. 17 upper column), appearing as additional peaks outside of the chromatin stained region in the intensity profile. In addition, at a later apoptotic stage the clear exclusion from the nucleoli in control cells vanished. None of theses changes in protein distribution could be detected using the monoclonal antibody specific for hnRNP A1, clone 4B10 (Fig. 17 lower column). Here, omission of nucleolar structures as well as the ring staining along the nuclear envelope was not changed after apoptosis induction.

These observations suggest that the change in protein distribution detected inside the cell is specific for the hnRNP A2/B1 protein, but not for hnRNP A1.

Figure 17: Heterogeneous ribonucleoprotein A2/B1 form granular structures in the cytosol upon apoptosis induction.

HeLa cells were treated with TRAIL [300 ng/ml]. At the indicated time points, cells were fixed with 4 % paraformaldehyde and immunocytochemical staining with antibodies specific for hnRNP A1/A2 and B1 (upper panel, scale bar = 10 µm) and specific for hnRNP A1 (lower panel, scale bar = 20 µm) were performed.

Chromatin was stained with H-33342. The figure shows representative confocal images. The localisation of hnRNP A1/A2/B1 and hnRNP A1 inside the cell is illustrated by comparing its distribution profile to the one of Hoechst, and is demonstrated for one representative cell.

Figure 18: Characterisation of hnRNP A2/B1 and A1 distribution in the cell-free reaction.

(A) Characterisation of hnRNP A1/A2/B1 distribution in isolated nuclei and apoptotic extracts by immunoblot.

1 x 10⁶ nuclei and cytosol extracted from 1 x 10⁶ cells were analysed by Western blotting using antibodies specific for hnRNP A1/A2/B1. (B) 5 x 10⁵ nuclei and cytosol extracted from 5 x 10⁵ cells were analysed by immunoblotting. The blot was probed with an antibody specific for hnRNP A1 (clone 4B10).

(C) Characterisation of hnRNP A1 distribution in nuclei and cytosolic extracts recovered from the in vitro reaction by Western blot analysis. The blot was probed with antibodies specific for hnRNP A1 (clone 4B10). (D) Characterisation of hnRNP A1/A2/B1 distribution in nuclei and cytosolic extracts recovered from the cell-free apoptosis reaction by Western blot analysis. The cell-free apoptosis reaction was performed in the presence or

In the next step, changes in protein distribution during the cell-free apoptotic reaction were examined by Western blot analysis using both hnRNP A antibodies.

Examination of cytosolic extracts before the in vitro reaction by probing a Western blot with an antibody specific for hnRNPA1 revealed a time dependent increase of the cytosolic concentration of hnRNP A1 in the course of apoptosis (Fig. 18A). hnRNP A1 was reported to be cleaved during apoptosis into three fragments with molecular weights of 32, 29 and 16 kDa (Brockstedt et al., 1998). A cleavage band of hnRNP A1 at 29 kDa in late apoptotic extracts was detected (Fig. 18A).

Further, the hnRNPA1 content of the cytosolic extract before and after the in vitro reaction was compared. As shown in Fig. 18B the hnRNP A1-specific increase observed in the apoptotic extract during apoptosis (Fig. 18A) could be detected in nuclear and cytosolic compartments after the cell-free reaction. The observation implies that during the cell-free incubation hnRNP A1 equilibrated between the cytosol and the nucleus to a certain degree.

Surprisingly, the apoptosis specific cleavage band could only be observed in late stage apoptotic extracts before and after the cell-free reaction but not in the nuclear fraction. Most likely this fragment exits the nucleus during apoptosis induction.

The same set of experiments was repeated using the general anti-hnRNP A antibody. First, the occurrence of hnRNP A variants in isolated nuclei as well as in control and apoptotic cytosolic extracts prior to the in vitro reaction was examined (Fig. 18C). Three hnRNP-specific signals were detected in nuclei corresponding to the three splicing isoforms hnRNP B1, A2 and A1 (Patry et al., 2003). In control and apoptotic extracts gained after 30 and 60 min after CD95-L treatment, two bands were detected migrating at a smaller molecular weight than hnRNP A1. These bands (indicated by asterisks) could not be assigned to any of the known hnRNP A variants. Since it is known that hnRNP A proteins are extensively modified by phosphorylation and poly(ADP-ribosyl)ation, these bands may correspond to post-translationally modified hnRNP A1 and A2 proteins. These bands were absent in late apoptotic extracts (180 min TRAIL). The signal corresponding to hnRNP A2 was detected in all cytosolic samples, with a significant increase 180 min after apoptosis induction.

In Fig. 18D nuclei and cytosolic extracts were analysed for the occurrence of hnRNP A variants after having been submitted to the cell-free apoptotic reaction. The hnRNP A2- and B1-specific signals were present in cytosolic samples recovered after the in vitro reaction, although they were absent in the same extracts prior to the reaction (compare Fig. 18D with 18C). This implies that a certain amount hnRNPs A2 and B1 equilibrate between nucleus and

cytosol during the cell-free incubation. In cytosolic extracts recovered from late stage apoptotic reactions a significant increase of hnRNP A1 was observed. This increase was partially inhibited by the pan caspase inhibitor zVAD-fmk. Nuclei recovered from late stage apoptotic reactions show only the specific signals for hnRNP A2 and B1.

These results are in accordance with the current knowledge that hnRNP variants shuttle between the nucleus and the cytoplasm to function as transporters for mRNA molecules out of the nucleus. The only way to traverse the nuclear membrane is through the nuclear pore complex, a macromolecular complex spanning the nuclear envelope. Small molecules (< 40 kDa) pass the pore by passive diffusion. For bigger molecules a complex and tightly regulated active transport process is necessary.

To evaluate if active transport between the cytosol and nucleus is important for the increase in hnRNP A1 observed in late stage apoptotic extracts recovered after the in vitro reaction, a cell-free apoptosis reaction in the presence of WGA (wheat germ agglutinin) was performed.

WGA blocks specifically the nuclear pore complex and is a commonly used inhibitor of active nuclear transport. Isolated mouse liver nuclei were incubated prior to the in vitro reaction with WGA for 10 min on ice. Two effects on the localisation of hnRNP A variants in apoptotic reactions but none in control reactions were observed and are shown in Fig. 18D.

Firstly, hnRNP A1 was retained in late apoptotic nuclei when nuclear transport is blocked;

secondly, the hnRNP A-specific signal which was observed in Fig. 18C (labelled by asterisks) was detectable in nuclei incubated with WGA.

Together, the effect of WGA on the distribution of hnRNP A1 in the cell-free apoptosis reaction (Fig. 18D) and the formation of hnRNP A2/B1 specific granular structures within the cytosol observed in immunocytochemical studies (Fig. 17) strongly suggest that the hnRNP A/B variants leave the nucleus during apoptosis.