M.Sc presentation: “Ultrastructural localization of RNase A and RNase L in cell models with different transcriptional activity”.

1. Ultrastructural localization of RNase A and RNase L in cell models with different transcriptional activity by Sara Cortesi Laboratory of Cell Biology and Neurobiology University of Pavia

3. PF, PG, IG Perichromatin fibrils (PF; +->), which are the in situ form of nascent transcripts at the edge of condensed chromatin Perichromatin granules (PG; ->), produced by folding of hnRNP containing perichromatin fibrils. Monneron and Benhard, 1969 Interchromatin granules (IG), storage site of inactive factors involved in transcription, for example splicing factors.

4. Balance in transcription Approximately 95% of hnRNA will never undergo translation. mRNA turnover performs an important role in regulation of gene expression. Jackson et al., 2000 These hnRNAs are degraded in the nucleus, even if they were correctly mature.

5. Ribonucleases RNase A , discovered in 1938, can digest internucleotide linkages in RNA molecules. RNase L is specifically induced in apoptosis and activated by an interferon-dipendent pathway. Ribó et al., 2006 Tanaka et al., 2004

6. Research Hypothesis and Approach The research hypothesis was that changes in the nuclear localization and quantities of RNases A and L provide important information concerning their roles in the regulation of transcription. Here I applied immunocytochemical (immunogold labelling of thin sections) and in situ hybridization techniques in cell and tissue models with different transcriptional activity, both physiologically and as experimentally induced. Statistical significance was determined by two-way ANOVA and signficance was set at P < 0.05. Primary antibodies Secondary antibodies Colloidal gold a-RNase A (rabbit; 1:200) Goat-Anti-Rabbit (1:20) 12 nm a-RNase L (mouse; 1:100) Goat-Anti-Mouse (1:20) 6 nm a-hybrid (rabbit; 1:400) Goat-Anti-Rabbit (1:20) 12 nm

7. I also investigated whether 2 peptides, ( DADL-Enkephalin (DADLE) and DAL-Enkephalin (DALE) ), known to downregulate transcription, modify RNase A levels by immunogold labelling in HeLa cells. DADLE treatment elicited decreased amounts of RNase A compared with controls and this effect was mantained after 48 h of recovery. DALE also evoked decreased RNase A but by contrast this was not mantained after recovery. RNase A

8. HeLa DADLE HeLa DADLE + REC Immunogold colocalization RNase A a-RNase A (12 nm) a-RNase L (6 nm)

9. I also measured RNAse A content in thin sections of thymus as a model of a tissue in which RNAse L becomes induced. RNase A quantity decreases in apoptotic thymocytes in comparison with non- apoptotic thymocytes. RNase A: regulation in thymus

10. RNase A levels increased in HeLa cells treated with heat shock (42 degC, 1 h) or 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole ( DRB ; a chemical blocker of the termination of transcription) if compared with control cells. RNase A: regulation in HeLa cells

11. HeLa (control) HeLa (Heat shock) Heat shock increases nuclear RNase A levels a-RNase A a-RNase L

12. Previous work from our laboratory had shown altered transcription in male mice fed with glyphosate-resistant genetically-modified (GM) soy. RNase A increased in spermatocytes and spermatids of mice fed for 2 months with modified soy containing diet (GM+). Feeding of GM soy modifies RNase A content in cells of mouse testis Sertoli Spermatocytes Spermatids GM- GM+ GM- GM+ GM- GM+ 2 months ++ ++ °° °°° ** *** 8 months ++ ++ °° °° ** **

13. 2 months control (natural soy) 2 months GM soy RNase A levels in spermatids of soy-fed mice a-RNase A a-RNase L

14. RNase L in Sertoli cells of soy-fed mice Sertoli cells of mice fed with transgenic soy (GM+) present an increased quantity in RNase L if compared with mice fed with a diet containing natural soy (GM-). Sertoli Spermatocytes Spermatids GM- GM+ GM- GM+ GM- GM+ 2mm ++ +++ °° °° ** ** 8mm ++ +++ °° °°° ** ***

15. Spermatocytes and spermatids from 8 month-old mice fed with a transgenic soy-containing (GM+) diet showed an increase in RNase L amount compared to those consuming a natural soy-containing diet (GM-). RNase L content in testicular cells of soy-fed mice

16. Apoptotic thymocyte In apoptotic thymocytes RNase L significantly increased in quantity in comparison with non- apoptotic cells. RNase L content increased in apoptotic thymocytes

17. Altered transcriptional activity in post-hepatectomy liver

18. (1) RNase A amount varies dependently on cell transcriptional activity and intensity of stimulus. Low intensity, long lasting stimuli (HeLa DADLE, HeLa DALE and thymocytes) cause decrease in enzyme amount. High intensity stimuli (HeLa heat shock, HeLa DRB, hepatocytes and germ line cells of 2 months old mice fed with GM soy) induce increase in RNase A quantity. (2) RNase L quantity increases only in apoptotic thymocytes and in 8 months old mice fed with GM soy. Conclusion: regulation of RNase levels in cells

19. Conclusion: RNase localization On perichromatin fibrils RNases probably work in a co-transcriptional degradative mechanism. RNases also bind interchromatin granules, in which they could be stored. They could leave these sites to reach PF and then come back to IG. RNases could also digest PF, that are often visualised at periphery of IG. In further work not presented here because of time constraints, I showed the following: (1) RNase A and RNase L bind perichromatin fibrils and interchromatin granules.

20. IG PF RNase PG