Messenger RNA maturation. Dr.ssa Mariangela Morlando

Transcription

1 Messenger RNA maturation Dr.ssa Mariangela Morlando

2 Features of RNA molecule Synthe(sed from a DNA template (transcrip(on) Single strand molecule Contains ribose in place of deoxyribose Contains Uracil in place of Thymine Types of RNA molecules Coding RNA which are translted into proteins: messenger RNA(mRNA) Non coding RNA which are never translated into protein but work as RNA with structural and/or regulatory func(on.

3 Messenger RNA

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5 mrna structures AUG UAA cap AAAAAAAAAAAAA 5 UTR 3 UTR ORF

6 Eucaryotic gene expression transcription START site exons upstream elements promoter elements TRANSCRIPTION CAPPING introns gene m 7 G pre-mrna m 7 G SPLICING POLYADENYLATION AAAAAAAAAn polyadenylated pre-mrna m 7 G TRANSPORT AAAAAAAAAn mature mrna TRANSLATION

7 Eucaryotic gene expression transcription START site exons upstream elements promoter elements TRANSCRIPTION CAPPING introns gene m 7 G pre-mrna m 7 G SPLICING POLYADENYLATION AAAAAAAAAn polyadenylated pre-mrna m 7 G TRANSPORT AAAAAAAAAn mature mrna TRANSLATION

8 Why the Cap structure is important? 1) RNA stability 2) Favours the mrna transport to the cytoplasm 3) Increases transla(on (it binds to eif4e that belongs to transla(on ini(a(on complex)

9 7-metilguanosina This involves the addi(on of a modified guanine base to the 50 end of the RNA. Specifically, it is a methylated guanine, and it is joined to the RNA transcript by an unusual 5 5 linkage involving three phosphates CAP is added at very early stage of transcrip(on ini(a(on estremità 5 della RNA catena The 5-5 phosphodiester bond makes the molecule resistant to the exonuclease ac(vity. I vitro synthe(zed RNA without CAP are rapidly degraded

10 pre-mrna capping The 5 cap is created in three enzyma(c steps: 1. a phosphate group is removed from the 5 end of the transcript. RNA triphosphatase 2. GMP moiety is added. RNA guanylyltransferase 3. GMP nucleo(de is modified by the addi(on of a methyl group. RNA(G-7-) methyltransferase

11 5 CAP favours the mrna transport to the cytoplasm CBC: CAP binding complex

12 Phosphorylation state of the CTD of RNA polymerase II along transcription cycle

13 DSIF/NELF-mediated checkpoint to ensure pre-mrna capping. TFIIH phosphorylate s the RNAP II CTD on Ser 5. DSIF interacts with RNAP II shortly after initiation. NELF recognizes the RNAP II DSIF complex and halts elongation. This pause allows the recruitment of the capping enzyme (CE) which adds a 5 -cap to the nascent transcript NELF is released by the concerted action of P-TEFb phosphorylation of DSIF and the CTD on Ser 2, PRMT1/5 methylation of Spt5, and the capping enzyme itself. FCP1 may also participate, as FCP1 is required to release the capping enzyme. The precise mechanism causing NELF release is unknown. Robert J. Sims III et al. Genes Dev. 2004;18:

14 Eucaryotic gene expression transcription START site exons upstream elements promoter elements TRANSCRIPTION CAPPING introns gene m 7 G pre-mrna m 7 G SPLICING POLYADENYLATION AAAAAAAAAn polyadenylated pre-mrna m 7 G TRANSPORT AAAAAAAAAn mature mrna TRANSLATION

15 What is the func<on of the polya tail? 1) RNA stability 2) Favours the mrna transport to the cytoplasm 3) Increases transla(on efficiency by favou(ng the loading of ribosomal 40S subunit 4) mrna 3 end forma(on allows efficient transcrip(on termina(on.

16 Looking for consenus sequences SEQUENCE ALLINEAMENT OF cdnas STARTING FROM THE POLYA TAIL 5 UTR 3 UTR nt globin AUG UAA GGUUAUCCAUCAAUAAA.GCUAUACGCAAAA n Ig AUG UAA CCACUGGGCCAAUAAA.GCUAUACGCAAAA n Ovoalbumin AUG UAA GCAACCUCGAAUAAA.GCUAUACGCAAAA n Polymerase AUG UAA AUCUGGAGGAAUAAA.GCUAUACGCAAAA n Myc AUG UAA UAUAGAUCCAAUAAA.GCUAUACGCAAAA n AAUAAA consensus

17 Efficiency consensus Polyadenyla(on efficiency Influence of consensus sequence AAUAAA!

18 Site- specific muta<on Recombinant DNA preparation * mutazione transfection mut1 mut2 mut3 WT polya Northern blot RNA extraction After 24 h HeLa cells

19 3 end forma(on in mammalian cells Cleavage and polyadenyla(on site 5 AAUAAA GU rich nt 5 AAUAAA OH P GU rich 3 5 AAUAAA A 250 degrada(on

20 SPECIFIC COMPLEXES ARE INVOLVED IN mrna 3 END PROCESSING Cleavage and Polyadenylation Specific Factor" CPSF! 5 AAUAAA sito di taglio CA Cleavage Stimulation Factor" CstF! " PAP 3 Poly- A polymerase GU- rich CFI! CFII! Curr Opin Cell Biol Jun;16(3):272-8"

21 band shift/emsa assay: to study RNAprotein interaction in vitro In vitro transcription Labelled RNA 2 Purified protein 1 Labelled RNA

22 band shift/emsa assay: to study RNAprotein interaction in vitro 1 Labelled RNA 2 Purified protein RNA-protein complex Free RNA

23 Cleavage and Polyadenylation Specific Factor" CPSF! 5 Cleavage Stimulation Factor" CstF! " PAP 3 AAUAAA Poly- A polymerase GU- rich sito di taglio CA CFI! SPECIFIC COMPLEXES ARE INVOLVED IN mrna 3 END PROCESSING CFII! HeLa NE EMSA assay for tes(ng the interac(on between CPSF 160 and consensus sequence AAUAAA AAUAAA AAGAAA Curr Opin Cell Biol Jun;16(3):272-8" complex sito di taglio free RNA

24 3 - End Forma<on: RNA Processing Cleavage and Polyadenylation Specific Factor Cleavage Factors Cleavage Stimulation Factor

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27 degrada<on 27

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29 29

30 CAP and polya tail influence efficient translation

31 3 end formation occurs during transcritpion

32 Polyadenylation is linked to termination mrna Torpedo Model AAAAAAAAA pre- mrna CPSF pa Xrn2 CstF RNAPII RNAPII

33 Polyadenylation is linked to termination Torpedo model Allosteric model

34 Variations at the transcriptome 3 end when processing gets alternative Approximately up to 70 % of the transcriptome is affected by a mechanism widely referred to as alternative 3 end cleavage and polyadenylation (APA) APA can be regulated on the level of mrna 3 end processing by various cis- and trans-acting determinants: (1) the intrinsic strength of sequence elements, (2) the concentration or activity of polyadenylation factors, and/or (3) tissue- or stage-specific regulatory factors are the most important key players

35 APA is highly regulated during development Proliferating cells tend to use upstream ( proximal ) PASs and produce mrnas with shorter 3 UTRs, while quiescent/differentiated cells favor downstream ( distal ) PASs and produce mrnas with longer 3 UTRs In contrast during somatic reprogramming or tumorigenesis, proximal PASs are favored leading to shorter 3 UTRs APA transcript isoforms of the same gene can encode different proteins and/or change the 3 UTR properties, leading to: - the inclusion or exclusion of mrna stabilizing or destabilizing elements, - mirna target sites, - result in different translation efficiencies - result in different subcellular localization

36 Alterna(ve polyadenyla(on of the immunoglobulin μ heavy chain gene sub obtimal pa obtimal pa Secreted terminus Membrane bound terminus Plasma cells B cells Secreted terminus Membrane bound terminus

37 CSTF-64 levels control the alternative processing of mrna. In not activated B cells the limiting concentration of CSTF allows the recognition of the stronger polyadenylation signal. The immuglobulin produced will then contain a portion for binding to the membrane. After the activation of B cells CSTF levels are increased and this allows t h e u s e o f t h e w e a k e r polyadenylation site that will be preferentially used because it will be the first to be transcribed. CstF-64 The polyadenylation factor CstF-64 regulates alternative processing of IgM heavy chain premrna during B cell differentiation. Takagaki Y, Seipelt RL, Peterson ML, Manley JL. Cell 1996 Nov 29;87(5):941-52

38 U1A: an example of on-off regulation of the cleavage and polyadenylation step in RNA processing U1A is a protein that binds to U1snRNA Obtimal U1A binding site

39 U1A is able to bind to its mrna The mrna encoding for U1A protein has a subop<mal polya site (AUUAAA) and 2 sequences similar to the U1A binding site on U1snRNA U1A - EMSA assay with increasing amount of U1A protein revealed that U1A binds its own mrna and that this lawer possessed two U1A binding sites RNA free

40 U1A autoregulation When the level of U1A exceeds that of U1 snrna, the excess U1A protein binds to its two binding sites in U1A pre-mrnas. Since U1A binding does not prevent cleavage of the pre-mrnas, free 3 ends are generated in the normal fashion In the absence of polyadenylation, however, both cleavage products are rapidly degraded by an exonuclease, so no functional U1A mrna is produced. As a result, synthesis of U1A protein is decreased until all the excess is used in formation of new U1 snrnps. Sub-obtimal U1A binding site sub-obtimal site

41 influenza A by its connection to the cellular 3 end apparatus has devised an efficient way to specifically shut off cellular gene expression The influenza A NS1 protein is one of the most abundant proteins synthesized in infected cells It interacts with the cellular 30 kda subunit of CPSF. By sequestering CPSF30 it prevents the binding of the CPSF complex to the RNA substrate thus inhibits 3 end cleavage and polyadenylation of the host pre-mrnas by preventing NS1 also targets PABPN1, which inhibits the processive synthesis of long poly(a) tails catalyzed by PAP As mrna processing represents a prerequisite for cytoplasmic export, the uncleaved host premrnas are retained in the nucleus, while viral RNAs are still exported.

42 APA in human disease - Loss of function mutations of globin mrna 3 end processing are a well recognized cause of thalassemias. Thalassemia patients have different mutations that result in an alteration of the AAUAAA hexanucleotide of the α globin and β globin genes, and invariably inactivate or severely inhibit gene expression. - Gain of function mutation stimulating 3 end processing in the prothrombin (coagulation factor II; F2) gene. The G A mutation converts the physiologically inefficient GC F2 cleavage site into the mechanistically most efficient CA dinucleotide, which increases cleavage site recognition and results in an enhancement of prothrombin mrna and protein expression. The result in an increased risk to develop thrombosis (referred to as thrombophilia). - defective PAP causes cell cycle arrest in the G 0 G 1 phases, the activity of PAP thus likely reflects the proliferative activity of cells è PAP mrna is overexpressed in human carcinomas of the breast, colon, ovary and pancreas and polyadenylation activity is significantly enhanced in aggressive acute leukemias and Burkitt lymphoma compared with less aggressive chronic leukemias and normal lymphocytes - mrnas with shorter 3 UTRs tend to be more stable or globally elevated eventually leading to higher protein output è switching to shorter 3 UTRs may allow proto-oncogenes to escape from inhibition by mirnas, thereby resulting in oncogene activation also in the absence of genetic alterations.

43 Histone mrna processing is polyadenylation independent

44 Common factors in RNA processing pathways! CPSF-73 endonucleolytic activity! Poly(A) + mrnas! Histone mrnas! CPSF-73 also displays 5 3 exoribonucleolytic activity that is responsible for the decay of the downstream cleavage product!