1. Purification and structural analysis of MALAT1 lncRNA Purification and Expression of the Telomerase RNA-Binding Domain Adam Biddlecome April 2, 2013 Professor Feigon Group Meeting 32 P

2. MALAT1 lncRNA Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) RNA is a nuclear localized lncRNA. It has been shown to have the 3’ A-rich end fold back and form a triple helix to stabilize the RNA. The triple helical form is largely protected from 3’-5’ exonucleases (Wilusz et al, G&D, 2012). Interesting characteristics include its apparent role as a translation enhancer and its overexpression in many human cancers.

3. MALAT PN and GD Constructs MALAT PNMALAT GD Brown et al, PNAS 2012 Wilusz et al, G&D 2012 The construct derived from the Genes and Development paper was largely the same except for a shorter upper stem and the addition of a UUCG tetraloop. The other construct had shorter upper and lower stems, while the GG bulge was maintained.

4. Test Transcription and Denaturing Gel Conditions chosen: 4 mM NTPs, 25 mM MgCl 2, 0.5 ml of Anneal Mix.

5. Native Gel of MALAT GD The construct at pH 6.4 consistently ran a little slower. At both pH 6 and pH 6.4 there are two bands.

6. MALAT PN Titration: Temperature

7. MALAT PN Varying Salt Conditions

8. MALAT GD Temperature

9. MALAT GD Magnesium

10. MALAT GD at different pH’s There is no downfield peak indicating a Hoogstein ‘C’ as would be seen in a triple helix.

11. MALAT GD NOESY A lot of overlap in the U-A region but still does not seem to be forming a triple helix.

12. UV Melting of MALAT GD

13. An in Trans MALAT Construct Hypothesized that a longer stem and an insert that could be annealed in trans to the construct would help achieve the desired triple helical structure. Insert-1: 5’ – AAA AAA AAA AAG CAA AA- 3’ Insert-2: 5’ - AAA AAA AAA AAG CAA AAG - 3‘

14. Test Transcription of Inserts and Trans Construct Insert 1 without an extra G at the 5’ end of the RNA sequence was unable to trancribe (insert 2 also transcribed at a relatively low rate).

15. MALAT Trans Gel Shift Assay It’s not a clear result, but there might be a slight shift up of the Trans construct band. Likely there is not binding seen and this is just an artifact. Insert 2 Insert is just 2 nM, 32 P- labeled

16. TRBD and CR4/5 The Medaka fish telomerase RNA has a CR4/5 region with documented specificity to TRBD. Bley et al. PNAS 2011

17. Induction Gel of TRBD Overnight induction at 37 °C of the MBP- TRBD fusion protein with IPTG. Purified with an amylose column. Wash Buffer (pH 7.5) 50 mM Tris-­‐HCl, 10% Glycerol, 0.5 M KCl, 5 mM beta-­‐mecaproethanol, 1 mM MgCl 2, 1 mM TCEP, 1 mM PMSF Elute Buffer (pH 7.5) 50 mM Tris-­‐HCl, 10% Glycerol, 0.5 M KCl, 5 mM beta-­‐mecaproethanol 1 mM MgCl 2, 1 mM TCEP, 1 mM PMSF, 10 mM maltose 80 kDa 60 kDa

18. Elute Profile and Concentrated TRBD Most of the decent yield of MBP-TRBD was recovered in the second elution. 50 kDa 80 kDa

19. CR4/5 RNA Gel Shift with MBP-TRBD The CR4/5 band thickness decreases and a small band is seen indicating RNA binding of the protein. MBP-TRBD

20. TRBD Factor Xa Enzyme Gel We see little to no difference in cutting with or without the CR4/5. MBP ~40 kDa TRBD ~32 kDa 80 kDa 50 kDa 30 kDa

21. Future Directions for Purification of TRBD Increase amounts of Factor Xa to see if more of the fusion protein can be cleaved. Try adding more calcium than is already present in the reaction buffer.

22. Special Thanks Yaqiang Wang Mahavir Singh Professor Juli Feigon