Biologia do RNA List of questions Transcription 1. How many types of RNA exist in the eukaryotic cell? What are their functions? 2. How many RNA polymerases are in the eukaryotic cell? What are their functions? Where are they localized in the cell? How can they be distinguished? 3. What is the function of RNA polymerase II? What are the main components and features of RNA polymerase II? 4. What is the “transcription cycle”? What are the main steps of RNA Polymerase II transcription? 5. What is the CTD? 6. How does the CTD phosphorylation pattern changes during the transcription cycle? What is its function? How does it work? 7. What are the main characteristics of a mature mRNA? 8. What is the 3’UTR? How does it regulate gene expression? 9. Comment the sentence: “An mRNA is never ‘naked’ in the cell” 10. What is an RNA binding protein? What is it main characteristic? Give one example of an RBP and describe its function.

RNA Processing: 5’ Capping and 3’ end processing 1. What is the 5´-CAP structure of mRNA? How is it synthesized? 2. What are the main functions of the CAP structure? 3. How is the 3’ end of tRNA formed? 4. How is the 3’ end of the histones pre-mRNA formed? 5. How is the 3’ end of the other pre-mRNAs (that code for proteins, with exception

of the histones) formed?

6. What are the functions of the polyA tail?

7. What is the polyadenylation signal? What does it do? 8. What is a strong polyadenylation signal? What is a weak polyadenylation signal? 9. What is a DSE signal? Describe the ‘architecture’ of a polyadenylation signal.

Indicate all the cis elements. 10. Where is the cleavage site located in relation to the main polyadenylation signal? 11. What is the CPSF complex? Where does it bind? What is its main function? 12. What is the CstF complex? Where does it bind? What is its main function? 13. What is the function of the polyA polymerase? 14. What is the function of the PolyA binding protein (PABP)? 15. Comment the sentence: “polyadenylation occurs in the nucleus and is co-

transcriptional”. 16. Comment the sentence: “Not all genes have a polyadenylation signal”. 17. Comment the sentence: “The AAUAAA signal is necessary but not sufficient for

polyadenylation”. 18. What are the consequences of mutations in the AAUAAA signal? 19. Comment the sentence: “A weak polyA signal is more prone to regulation” 20. What is the function of the USE signal in polyadenylation? Does it always exist? 21. Pre-mRNA 3´-end formation can serve as a mechanism for auto-regulate mRNA

levels. How? Describe an example. 22. How can defective 3´-end pre-mRNA processing cause disease? Give an example. 23. What is alternative polyadenylation? What is its function? How can it be

regulated?

24. Why is the concentration of cleavage and polyadenylation factors so critical during alternative polyadenylation?

25. Comment the sentence: “alternative polyadenylation is co-transcriptional and

depends on the speed of transcription”

26. Give an example of how alternative polyadenylation affect gene expression. 27. Does alternative polyadenylation define the 3’UTR length? How? How does this

affect gene expression? In which cellular conditions this may happen? 28. Comment the sentence: “Formation of mRNA 3´-ends in eukaryotes is regulated

and interrelated with other steps in mRNA synthesis?

Splicing 1. What is an intron? Define the concept of splicing in general terms? 2. What is a splicing signal? Describe the various elements of the splicing signal? 3. What is a strong splicing signal? How does it differ from a weak signal? 4. What is the spliceosome? What does it do? 5. What is a snRNP? How many snRNPs there are? Describe its components?

6. What is the function of U snRNAs in splicing? 7. Comment the sentence: “The spliceosome is a molecular machine”. 8. What is the function of the splicing factors non-snRNP? 9. Describe the mechanism of splice site selection? 10. What is an ESE? What is a ESS? What is an ISE? What is an ISS?

11. What is the function(s) of the SR proteins in splicing? 12. Describe the Spliceosome assembly cycle? 13. Why does altered splicing cause disease? 14. What is alternative splicing? What is its main function? 15. How does alternative splicing switch off gene expression? 16. How does a cell choose a splice site during alternative splicing?

17. What is the function of specific histone “marks”, in particular H3 acetylation in

alternative splicing? How does it work?

18. What are alternative splicing regulators? Give examples of each type? 19. Why is the concentration of splicing factors so critical during alternative

splicing? 20. Comment the sentence: “alternative splicing is co-transcriptional and

sometimes depends on speed of transcription”. 21. Comment the sentence: “Identical pre-mRNAs are differentially spliced in

different organs”. 22. Comment the sentence: “In vertebrates, in particular in humans, the number of

proteins is much higher than the number of genes”.

Integration of co-transcriptional events 1. What is the “mRNA factory model”?

2. How is transcription by RNA polymerase II interconnected with pre-mRNA 5’capping? 3. How is transcription by RNA polymerase II interconnected with splicing? 4. How is transcription by RNA polymerase II interconnected with polyadenylation? What are the polyadenylation factors that are recruited to RNA polymerase II in the promoter?

Methods 1. How would you measure mRNA steady state levels in a sample with a small amount

of RNA?

2. Name a method to analyse the binding pattern of a specific protein to chromatin and describe it briefly.

3. Name a method to analyse the sequences on the RNA that bind to a specific protein?

4. Name a method to identify the mRNAs that a protein bind to?