RNA BIOLOGY 2007-12-15 (Manuel Santos) Exam preparation: The 5and 3 ends processing of Pre-mRNA

1. O que a estrutura 5-CAP do m RNA? Como sintetizada? A estrutura 5CAP pode ser encontrada na extremidade 5 da molecula de RNA e consiste num nucleotido de guanina ligado ao mRNA atraves de uma ligao trifosfatidica. Esta guanosina metilada na setima posio por uma metil transferase e designa-se por 7-metilguanosina cap
2. 3. Further modifications include the possible methylation of the 2' hydroxy-groups of the first 3 ribose sugars of the 5' end of the mRNA. The methylation of both 2' hydroxy-groups is shown on the diagram. Functionally the 5' cap looks like the 3' end of an RNA molecule (the 5' carbon of the cap ribose is bonded, and the 3' unbonded). This provides significant resistance to 5' exonucleases.

The starting point is the unaltered 5' end of an RNA molecule. This features a final nucleotide followed by three phosphate groups attached to the 5' carbon. 1. One of the terminal phosphate groups is removed (by a phosphatase), leaving two terminal phosphates. 2. GTP is added to the terminal phosphates (by a guanylyl transferase), losing two phosphate groups (from the GTP) in the process. This results in the 5' to 5' triphosphate linkage. 3. The 7-Nitrogen of guanine is methylated (by a methyl transferase). 4. Other methyltransferases are optionally used to carry out methylation of 5' proximal nucleotides.

Para ocorrer formao da CAP um fosfato da extremidade 5 do RNA deve ser

liberado por hidrlise, gerando uma extremidade 5 difosfato, Essa extremidade difosfato deve ento se ligar a um tomo de fsforo de um GTP, gerando uma ligao incomum 5-5 trifosfato. O nitrognio que se encontra na posio 7 da guanina da extremidade deve ento sofrer uma metilao, mediada pela enzima S-adenosil-metionina, formando o chamado cap 0. As riboses dos dois primeiros nucleotdeos do RNA podem tambm ser metiladas na extremidade 2'-OH, formando os chamados cap 1 e cap 2.

4. Quais as funes principais da estrutura CAP?

 uma modificao que ocorre na extremidade 5 e que confere molecula de mRNA maior estabilidade, protge-a da aco de fosfatases e nucleases e aumenta a probabilidade desse RNA ser capturado pelos ribossomas para traduo, esta estrutura no est presente no tRNA e no rRNA acontecendo principalmente nos mRNA e hnRNA.
1. 2. 3. 4. Regulation of nuclear export. Prevention of degradation by exonucleases. Promotion of translation (see ribosome and translation). Promotion of 5' proximal intron excision.

3. O que a poliadenilao do pre-mRNA? a adio de uma cauda de poliadenilato ao mRNA na sua extremidade 3, esta cauda composta por mltiplas adenosinas monofosfatadas (s com adeninas), tem a funo de actuar como acentuadora da traduo, proteger o mRNA da digesto por nucleases presentes no meio e proporcionar uma maior estabilidade molcula. Especula-se que ela tambm tenha um papel importante no transporte do mRNA para o citoplasma. interessante notar que o nucleotdeo que se encontra logo antes da cauda poliA no o ltimo que ser traduzido e podem haver mais centenas de nucleotdeos alm da extremidade 3 de um RNA maduro. 4. O que o sinal de poliadenilao? Para que serve? O mRNA clivados atravs de uma endonuclease especfica que reconhece a seqncia AAUAAA, no entanto, apenas essa seqncia no suficiente para que ocorra o corte, tambm necessrio que haja um contexto ainda desconhecido de nucleotdeos prximos que caracterizem a regio. Aps o corte, uma enzima polimerase poli-A, dependente de ATP, acrescenta os nucleotdeos contendo a base adenina na extremidade 3 do RNA. Serve para acentuadora da traduo, proteger o mRNA da digesto por nucleases presentes no meio e proporcionar uma maior estabilidade molcula.

5. O que um forte sinal de poliadenilao? O que um sinal fraco de poliadenilao? Um sinal forte de poliadenilao a sequencia AAUAAA (NNUANA). Um sinal fraco de poliadenilao a sequencia AAUAAG 6. O que um sinal DSE? Descreve o sinal de poliadenilao. Indica os elementos cis. Dois sinais directos indicam RNA polymerase II para parar a transcrio: os sinais localizados junto polyA e um elemento adicional a jusante (DSEdownstream element) localizado na regio de termino. O sinal de DSE (135pb) actua pausando a elongao da polymerase. 7. Quais so as funes mais importantes da poliadenilao do mRNA? A poliadenilao do mRNA adiciona a este uma cauda que protg a molecula de mRNA de degradao enzimatica, adiciona uma terminao transcripcional, ajuda na translao do mRNA do nucleo para o citoplasma. Mecanismo da poliadenilao: A maquinaria da poliadenilao no nucleo dos eucariotas trabalha em productos da RNA polymerase II como o mRNA. Neste um complexo de multi-proteinas CPSF: cleavage/polyadenylation specificity factor CstF: cleavage stimulation factor PAP: polyadenylate polymerase PAB2: polyadenylate binding protein 2 CFI: cleavage factor I CFII: cleavage factor II

Cliva a maior parte do RNA recentemente produzido junto extremidade 3 e poliadenila o final produzido por esta clivagem. A civagem catalizada pela enzima CPSF e ocorre cerca de 10-30 nucleotidos a jusante do sitio de ligao, geralmente designado pela sequencia de AAUAAA embora existam algumas variantes que se ligam mais fracamente ao CPSF). Duas outras proteinas especificas em se ligarem ao RNA so a CstF e a CFI. A Cstf liga-se a uma regio rica em GU a jusante da ligao (2 regiao de ligao) do CPSF e a CFI reconhece um terceiro sitio de ligao no RNA ( a sequencia UGUAA nos mamiferos) e recruta a CPSF mesmo que a sequencia AAUAA no exista. O RNA clivado apos a trasncrio e o CstF liga-se RNA polymerase II, esta clivagem envolve a proteina CFII. Quando o RNA clivado a poliadenliao comea catalisada pela poliadenilato polymerase que construi uma cauda PoliA adicionando unidades de adenosina monofosfatada ao RNA clivando o pirofosfato, a proteina PAB2 ligase nova cauda PoliA e aumenta a afinidade da poliadenilato polymerase para o RNA. Quando a cauda tem aproximadamente 250 nucleotidos a enzima no se consegue ligar mais ao complexo CPSF e a poliadenilao pra. Esta maquinaria tambem est ligada fisicamente ao spliceossoma (um complexo que remove intres do RNA) 8. Onde se situa o sitio de clivagem em relao ao maior sinal de poliadenilao? Entre 15 e 30 nucleotideos frente do sinal AAUAAA 9. O que o complexo CPSF? Como formado e qual a sua principal funo? CPSF Cleavage and polyadenylation specificity factor Est envolvido na clivagem do pr-mRNA sendo a primeira proteina a ligar-se junto zona de sinalizao ( sua esquerda a AAUAAA) qual a cauda de poliadenilao se vai ligar atraves da poliadenina polymerase. um complexo proteico formado por quarto proteinas, a CPSF-30, a CPSF-73, a CPSF-100 e a CPSF-160. A CPSF-73 a endonuclease que cliva o mRNA precursor, a ligao da poliadenina polymerase um requesito para ocorrer a clivagem. 10. O que o complexo CstF? Aonde se vai ligar? Qual a sua principal funo?

CstF - Cleavage stimulation factor uma proteina heteromerica com cerca de 200 kd que est envolvida na clivagem da regio 3 sinalizada de uma molecula de mRNA. Esta proteina recrutada pela CPSF e contem um complexo proteico que monta a sintese functional de uma cauda de poliadenilao da qual resulta uma molecula de RNA Madura para exportar do nucleo para o citoplasma. Este factor dependente das fases do ciclo cellular estando significativamente aumentado na transio da fase G0 para a fase S. 11. Qual a funo da polyA polimerase? Que tipo de Poly A polimerases existem? A funo da poliA polymerase catalizar a adio de adenosina ao terminal 3 do RNA numa seuquencia de forma independente recorrendo ao uso de ATP e produzindo pirofosfato e um residuo nucleotidico mais longo. Estas enzimas pertencem familia das transferases, ou seja enzimas que transferem grupos nucleotideos que contm fosforo. Existem as Poly(A) polymerase, Poly(A) synthetase, Polyadenylate nucleotidyltransferase, Polyadenylate polymerase, Polyadenylate synthetase, Polyadenylic acid polymerase, Polyadenylic polymerase

12. Como so formadas as primeiras 10 As formadas durante a poliadenilao? Indica os factores cis e trans envolvidos. Estes dependem do sinal da sequencia inicial e a sua adio ocorre de forma lenta 13. O que acontece aps a adio das primeiras 10 As? Descreve o mecanismo da elongao da poliA. A elongao independente do sinal da sequencia inicial, depende da oligoA adicionada durante a inicio, a adio de bases de A rapida (mais 200) e requer o factor PABPN1 ou PABPII (poly-adenine binding protein II) que aumenta o processamento da poliA polymerase. 14. Como a poliA degradada? Indica as enzimas envolvidas no encurtamento da

cauda poliA? A deadenilao e degradao pode ocorre no citoplasma, em mamiferos, a poliA ribonuclease PARN pode-se ligar 5CAP e remover nucleotidos da cauda poliA. O nivel de acesso 5CAP e cauda poliA importante no controlo do tempo de degradao do mRNA. A PARN pode remover menos As se o RNA estiver ligado aos factores de inicio 4E no 5CAP e ao 4G na cauda poliA, desta forma a translao reduz a eliminao de bases. O racio de deadenilao pode ser regulado por proteinas com especificidade para o RNA, assim que a cauda poliA removida o complexo de deccaping remove a 5CAP e degrada o RNA 15. O que acontece quando a cauda poliA do mRNA removida ou degradada? As caudas de PoliA no citoplasma so encurtadas gradualmente e os mRNAs com caudas mais pequenas so traduzidos menos vezes e degradados mais depressa. 16. O que um elemento cis de nome ARE? Uma sequncia rica em adeninase uracilos(ARE)localizada 60nt a montante da cadeia de poli(A) confere o controlo. 17. Descreve a maturao citoplasmatica da cauda poli A? Indica os elementos cis e trans que participam neste processo biologico?

18. Qual a funo da proteina de ligao da poliA PABP? uma proteina que se liga cauda da poliA, a sua isoforma nuclear liga-se selectivamente a cerca de 50 nucleotidos e estimula a actividade da poliA polymerase. 19. Comente a frase: a poliadenilao ocorre no nucleo e cotranscricional. A poliadenilao ocorre no nucleo e sem ela o RNA seria degradado no citoplasma portanto pode-se considerar cotranscicional. 20. Comente a frase: Nem todos os genes possuem um sinal de poliadenilao.

21. Comente a frase O sinal AAUAAA necessario mas no suficiente para a poliadenilao. 22. Quais as consequencias de mutaes no sinal AAUAAA As consequencias podem resultar num sinal mais fraco ou num sinal irreconhecivel. 23. Comente a frase: Um sinal de poliA fraco mais propenso regulao. 24. What is the function of the USE signal in polyadenylation? Does it always exist? 25. As histonas pre-mRNA no so poliadeniladas. Porque? Estas acabam num stem-loop seguido de uma sequencia rica em purinas (cerca de 20 nucleotidos) chamada de histone downstream element HDE que dirige onde o RNA cortado de modo a que a finalizao das histonas se forme. 26. Pre-mRNA 3-end formation can be regulated. How? Describe an example? 27. How can defective 3-end pre-mRNA processing cause disease? 28. What is alternative polyadenylation? What is its function? Uma vez que a poliadenilao alternativa altera o comprimento da 3UTR pode alterar os sitios de ligao para os microRNA que esta regio pode ter. Os microRNA tm tendencia a reprimir a translao e promovem a degradao dos mRNAs a que se ligam, embora existam alguns que estabilizem a transcrio. A poliadenilao alternativa pode tambem encurtar a regiao codificada o que pode fazer um mRNA para uma proteina diferente embora seja raro. Ou seja poliadenila em sitios diferentes?? 29. Comment the sentence: Formation of mRNA 3-ends in eukaryotes is regulated and interrelated with other steps in mRNA synthesis?

30. Comment the sentence: Transcription and pre-mRNA processing are coordinated processes. RNA BIOLOGY 2007 (Manuel Santos) Exam preparation: mRNA translation. 1. Describe in general terms the mechanism of 5-UTR scanning? 2. What is the role of the context of the AUG initiation codon? 3. What is the effect of mRNA secondary structure on 5-UTR scanning? 4. What is the function of eIF1, eIF1A and eIF2 in translation initiation? 5. What is the function of eIF3 in translation initiation? And eIF4?, eIF5? and eIF6? 6. Describe the composition of the 40S-eIFs complex during the initial stages of scanning? 7. What is the main function of eIF4E? what is eIF4F? describe its composition and how it activates mRNA translation? 8. What is the main function of the ternary complex? 9. What is the role of GTP hydrolysis during the initiation phase of mRNA translation? 10. How is GDP recycled on the eIF2 subunit? 11. Which eIF is an helicase? What is its function during translation? 12. What is the functional relevance of eIF2 phosphorylation by HRI, PKR, PERK and GNC2 kinases? 13. eIF4G is called an adaptor initiation factor? Why? Describe its function in CAP and non-CAP mediated initiation? 14. What is an IRES? How does it work? 15. Why is the polyA binding protein (PABP) relevant for translation initiation? 16. Some mRNA are not capped but still translate efficiently. Explain how is this possible? 17. Describe all types of alternative translation initiation? 18. What is an uORF? What is its main function? And when is it used?

19. Why do translation initiaton defects cause disease? 20. Comment the sentence: Cells respond to stress by reprogramming gene expression at the translational level 21. Comment the sentence: Virus reprogram gene expression of the host cell at the translational level. 22. mRNA translation defects causes neurodegenerative disease. How? 23. Comment the sentence: the 5-UTR is an important control point of mRNA translation 24. Comment the sentence: The interaction of the 5-UTR and the 3-UTR is important for translation efficiency. 25. How do the ends of mRNAs interact? RNA BIOLOGY 2007 (Manuel Santos) Exam preparation: Splicing 1. O que um intro? Define o conceito de splicing nos termos gerais? Um intro uma zona do gene no codificada. O splicing apenas ocorre em celulas eucarioticas e remove os intres juntando os exes durante a transcrio. A estrutura que cliva essas ligaes nucleotidicas o spliceossoma. 2. O que um sinal de splicing? Descreve os varios elementos de um sinal de splicing? A maioria dos intres comea com GU - dador de splice - e acaba com AG splice acceptor site. A sequencia de consenso na extremidade 5 AGGUAAGU e na extremidade 3 so 10 C ou U, seguidos de uma base qualquer, de um C e de AG. O splicing ocorre em duas estapas: duas transesterificaes. A transesterificao a transferncia da ligao fosfodiester. A primeira reao ocorre pelo ataque nucleoflico da adenina do stio de ramificao Guanina no stio de splice 5'.

Com a transferncia da ligao fosfodister, formada uma estrutura de ala. A segunda reao se d pela ligao de um xon com o outro, soltando assim o ntron, que ser degradado e reciclado na clula.

3. What is a strong splicing signal? How does it differ from a weak signal? As sequencias GU e AG no so suficientes para se denotar a presena de um intro, outra sequencia importante o branch site localizado 20 a 50 bases frente do sitio de aceitador. A sequencia de consenso deste sitio a CU(A/G)A(C/U) onde A conservado em todos os genes. 4. O que um spliceossoma? Qual a sua funo? Um spliceossoma um complexo que remove introes de mRNA transcrito, composto por 5 pequenas snRNPs ou seja pequenas unidades proteicas de RNA nuclear, que so a U1, U2, U4, U5 e a U6 que participam em diversas interaces entre RNA-RNA ou RNA-proteina. O RNA que as compoe rico em U. - Assegura a conservao da sequencia reconhecendo os splicing sites - Preciso nucleotidica na juno de exes - Condensa os exes (informao importante) 5. O que um snurp? Quantos snurps existem? Descreve os seus components. So as subunidades do spliceossoma ou snRNPs small nuclear RNA proteins, ou seja pequenas unidades proteicas de RNA nuclear, que so a U1, U2, U4, U5 e a U6 que participam em diversas interaces entre RNA-RNA ou RNA-proteina. 6. Comenta a frase: O spliceossoma uma maquina molecular. 7. Qual a funo dos factores de splicing no-snRNP?

E Complex-U1 binds to the GU sequence at the 5' splice site, along with accessory proteins/enzymes ASF/SF2, U2AF (binds at the Py-AG site), SF1/BBP (BBP=Branch Binding Protein);

A Complex-U2 binds to the branch site and ATP is hydrolyzed; B1 Complex-U5/U4/U6 trimer binds, and the U5 binds exons at the 5' site, with U6 binding to U2; B2 Complex-U1 is released, U5 shifts from exon to intron and the U6 binds at the 5' splice site; C1 Complex-U4 is released, U6/U2 catalyzes transesterification, that make 5'end of introns ligate to the A on intron and from a lariat ,U5 binds exon at 3' splice site, and the 5' site is cleaved, resulting in the formation of the lariat;

C2 Complex-U2/U5/U6 remain bound to the lariat, and the 3' site is cleaved and exons are ligated using ATP hydrolysis. The spliced RNA is released and the lariat debranches.

8. Describe the mechanism of splice site selection?

The model for formation of the spliceosome active site involves an ordered, stepwise assembly of discrete snRNP particles on the hnRNA substrate. The first recognition of hnRNAs involves U1 snRNP binding to the 5' end splice site of the hnRNA and other non-snRNP associated factors to form the commitment complex, or early (E) complex in mammals. [8][9] The commitment complex is an ATPindependent complex that commits the hnRNA to the splicing pathway. [10] U2 snRNP is recruited to the branch region through interactions with the E complex component U2AF (U2 snRNP auxiliary factor) and possibly U1 snRNP. In an ATP-dependent reaction, U2 snRNP becomes tightly associated with the branch point sequence (BPS) to form complex A. A duplex formed between U2 snRNP and the hnRNA branch region bulges out the branch adenosine specifying it as the nucleophile for the first transesterification.[11] The presence of a pseudouridine residue in U2 snRNA, nearly opposite of the branch site, results in an altered conformation of the RNA-RNA duplex upon the U2 snRNP binding. Specifically, the altered structure of the duplex induced by the pseudouridine places the 2' OH of the bulged adenosine in a favorable position for the first step of splicing.[12] The U4/U5/U6 tri-snRNP (see Figure 1) is recruited to the assembling spliceosome to form complex B, and following several rearrangements, complex C (the spliceosome) is activated for catalysis.[13][14] It is unclear how the triple snRNP is recruited to complex A, but this process may be mediated through protein-protein interactions and/or base pairing interactions between U2 snRNA and U6 snRNA. The U5 snRNP interacts with sequences at the 5' and 3' splice sites via the invariant loop of U5 snRNA[15] and U5 protein components interact with the 3' splice site region.[16] Upon recruitment of the triple snRNP, several RNA-RNA rearrangements precede the first catalytic step and further rearrangements occur in the catalytically active spliceosome. Several of the RNA-RNA interactions are mutually exclusive; however, it is not known what triggers these interactions, nor the order of these rearrangements. The first rearrangement is probably the displacement of U1 snRNP from the 5' splice site and formation of a U6 snRNA interaction. It is known that U1 snRNP is only weakly associated with fully formed spliceosomes[17], and U1 snRNP is inhibitory to the formation of a U6-5' splice site interaction on a model of substrate oligonucleotide containing a short 5' exon and 5' splice site.[18] Binding of U2 snRNP to the branch point sequence (BPS) is one example of an RNA-RNA interaction displacing a protein-RNA interaction. Upon recruitment of U2 snRNP, the branch binding

protein SF1 in the commitment complex is displaced since the binding site of U2 snRNA and SF1 are mutually exclusive events. Within the U2 snRNA, there are other mutually exclusive rearrangements that occur between competing conformations. For example, in the active form, stem loop IIa is favored; in the inactive form a mutually exclusive interaction between the loop and a downstream sequence predominates. [14] It is unclear how U4 is displaced from U6 snRNAm, although RNA has been implicated in spliceosome assembly, and may function to unwind U4/U6 and promote the formation of a U2/U6 snRNA interaction. The interactions of U4/U6 stem loops I and II dissociate and the freed stem loop II region of U6 folds on itself to form an intramolecular stem loop and U4 is no longer required in further spliceosome assembly. The freed stem loop I region of U6 base pairs with U2 snRNA forming the U2/U6 helix I. However, the helix I structure is mutually exclusive with the 3' half of an internal 5' stem loop region of U2 snRNA.

Mechanism of Splicing

U1 snRNP binds specifically to the 5 splicing site sequences in the RNA and also binds to conserved residues on the 3 splice site U2 snRNP binds to the branch point , helped by the U2 snRNP auxiliary factor (U2AF). (U2AF binds to the polypyrimidine U-C rich region adjacent to the 3 splice site and positions the U2 snRNP on the branch point.)

Tri-snRNP complex (U4+U5+U6) joins in. The 5 splice site has an adjacent conserved sequenceof exon to which the U5snRNA binds, forming the spliceosome sites where the catalysis can occur

For initiation, nucleophilic substitution occurs at the 5 splice junction by the 2-hydroxyl group of the branch point adenosine. Pairimg between U1 and intromn sequence is destabilized and U5 SnRNA bonds more strongly with 5' site Bond between U4 and U6 atrophies and U6 now bonds with both 5'site and branch point U1 (used for identification of 5' splice site) and U4 quits(needed for bringing U6 to catalytic site) Newly freed 5'exon replaces the 3' splice junction y by 3'OH group U5 retains contact with 5'end and free exon but also contacts with 3' splice junction Eventually the introns are removed and the exons form the mature mRNA by larhiat structure formation

9. What is an ESE? What is a ESS? What is an ISE? What is an ISS? 10. Qual a funo das proteinas SR no splicing? Estas proteinas so ricas em serina e arginina e esto envolvidas na regulao e seleco dos sitios propicios a splicing no mRNA. Tambem o splicing alternativo requer estas proteinas uma vez que seleccionam os sitios alternativos de splicing a usar. 11. Describe the Spliceosome assembly cycle?

12. Quanto spliceossomas existem nos eucariotas? Quais as principais diferenas entre eles? Existem 3 tipos de spliceossoma: O maior que divide os introes com GU a 5 e AG a 3, composto pelos snRNPs U1, U2, U4, U5 e U6 e est activo no nucleo. O menor que similar ao maio embora remova introes raros com sequencias de splice diferentes, composto por snRNP U5, U11, U12, U4atac e U6atac, pode ser encontrado no nucleo. Trans-splicing que uma forma de splicing que junta dois exoes que no esto no mesmo RNA a ser transcrito. 13. Porque que alteraes no splicing causam doenas? Alteraes no splicing implicam alteraes nas proteinas traduzidas, erros comum de splicing passam pela perda de funo de um sitio de splice resultando na exposio permatura de um codao de stop, perda de um exo ou incluso de um intro; mutao de um sitio de splice reduzindo a sua especificidade causando delees ou inseres de aminoacidos,; incluso ou excluso de mais RNA que o esperado resultando em exes maiores ou mais pequenos. 14. What is alternative splicing? What is its main function? Splicing alternativo a recombinao de diferentes exoes, tem como funo possibilitar uma maior diversidade genetica nos eucariotas. 15. Does alternative splicing exist in all eukaryotes? Where is it prevalent?w 16. How does alternative splicing switch off gene expression? 17. How does a cell choose a splice site during alternative splicing? 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. What is the exon junction complex (EJC)? What does it do?

22. Why is the EJC relevant to mRNA nonsense mediated decay? 23. Comment the sentence splicing is an ancient biological process. If you agree with the sentence describe the molecular evidence for early evolution of splicing? 24. Comment the sentence: Identical pre-mRNAs are differentially spliced in different organs. 25. Comment the sentence: In vertebrates, in particular in humans, the number of proteins is much higher than the number of genes. RNA BIOLOGY 2007 (Manuel Santos) Exam preparation: mRNA stability and Decay. 1. What are the main functions of the CAP and polyA in mRNA stability? 2. What is a premature termination codon? 3. Describe in general terms the concept on mRNA quality control, referring to mRNA surveillance mechanisms? 4. What is the exosome? How does it work? And how is it constituted? 5. Describe the mechanism of 5to 3 mRNA degradation? Identify the ribonucleases involved in this process. 6. What is mRNA decapping? What is its main function? 7. What is mRNA deadenylation? What is its main function? 8. Why is the interaction between 5and 3 mRNA ends relevant for mRNA stability? 9. How do the 5 and 3 ends of mRNA interact? How does the interaction becomes disrupted? What happens when it is disrupted? 10. What is an Exon Junction complex (EJC)? What is its function (s)? 11. What is NMD? Describe in general terms how it works? 12. What is the functional relevance of NMD? 13. Describe the roles of UPF1, UPF2 and UPF3 in NMD?

14. What is the role of the eRFs in NMD? 15. How does the NMD machinery recognize a premature termination codon (PTC)? 16. Explain how the iron metabolism is regulated at the translational level? 17. What is an ARE and how does it work?

RNA BIOLOGY 2007 (Manuel Santos) Exam preparation: Biology of microRNAs.

1. o que o microRNA (miRNA)?
MicroRNAs (miRNAs) are short ribonucleic acid (RNA) molecules, on average only 22 nucleotides long and are found in all eukaryotic cells. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression and gene silencing.[1][2] The human genome may encode over 1000 miRNAs,[3][4] which may target about 60% of mammalian genes[5] and are abundant in many human cell types.[6] miRNAs show very different characteristics between plants and metazoans. In plants the miRNA complementarity to its mRNA target is nearly perfect, with no or few mismatched bases. In metazoans on the other hand miRNA complementarity is far from perfect and one miRNA can target many different sites on the same mRNA or on many different mRNAs. Another difference is the location of target sites on mRNAs. In metazoans the miRNA target sites are in the three prime untranslated regions (3'UTR) of the mRNA. In plants targets can be located in the 3' UTR but are more often in the coding region itself.[7] MiRNAs are well conserved in eukaryotic organism and are thought to be a vital and evolutionary ancient component of genetic regulation.[8][9][10][11]

2. Describe the biogenesis of miRNAs and siRNAs in detail? 3. What are the main differences between Pri-mRNAs, pre-miRNAs and mature miRNAs? 4. What is the main function of miRNAs and siRNAs? Os miRNAs regulam a expresso gnica regulando a quantidade de proteinas produzidas por genes eucarioticos. Os siRNA defendem e ajudam a proteger a integridade do genoma. Estes RNAs de interferencia inibem a produo de virus e impedem a disperso de elementos de trasnposio para outros loci cromossomicos. 5. What is the main difference between miRNAs and siRNAs? Os miRNAs regulama expresso gnica e os siRNAs defendem o genoma

6. What is the main function of the Drosha complex? 7. What is the function of Dicer? 8. What is the RISC complex? What does it do? 9. What is the main function of argonaut proteins? 10. How do miRNAs control gene expression? Describe the mechanism in detail. 11. What is a stress granule? What is a P-body? What are the main differences between Stress granules and P-bodies? 12. Describe the main function of P-bodies? 13. How are P-bodies linked to the NMD pathway? 14. Comment the sentence: miRNAs are repressors of gene expression. 15. Why are miRNAs relevant to cancer biology? Describe a general mechanism of cancer development associated to miRNA biology. 16. Comment the sentence: miRNAs are involved development, differentiation, cell proliferation and many human diseases. 17. What is a miRNA expression profile? What is it used for? RNA BIOLOGY 17. What is a miRNA expression profile? What is it used for?