UC Biologia Molecular Past Paper PDF 2024-2025

UC Biologia Molecular (40395)| Ano lectivo 24_25 Parte II| Aula: 2 19 e 20 novembro SUMÁRIO Tipos e estrutura do RNA. Pre-mRNA splicing e processamento. Tipos de splicing e mecanismos de splicing do pre- mRNA. Tipos/grupos de intrões. Assemblagem e ciclo do spliceossoma. Associação do splicing com...

UC Biologia Molecular (40395)| Ano lectivo 24_25 Parte II| Aula: 2 19 e 20 novembro SUMÁRIO Tipos e estrutura do RNA. Pre-mRNA splicing e processamento. Tipos de splicing e mecanismos de splicing do pre- mRNA. Tipos/grupos de intrões. Assemblagem e ciclo do spliceossoma. Associação do splicing com as diferentes etapas da expressão de um gene. Locais de splicing. Impacto dos intrões na expressão de genes. Importância dos intrões na evolução de novas proteínas. Trans-splicing em tripanosomas e nemátodes. Splicing de tRNAs. Produção de rRNAs. Os terminais 3´ de mRNAs- poliadenilação. Types and structure of RNA. Pre-mRNA splicing and processing. Types of splicing and pre-mRNA splicing mechanisms. Intron types/groups. Spliceosome assembly and cycle. Association of splicing with the different stages of gene expression. Splice sites. Impact of introns on gene expression. Importance of introns in the evolution of new proteins. Trans-splicing in trypanosomes and nematodes. tRNA splicing. Production of rRNAs. The 3' ends of mRNAs- polyadenylation. 1 S. Mendo| BM24_25  When we talk about gene expression… ✓The spliceosome is a molecular machine, which together with RNA polymerases and ribosomes plays a critical role in basic gene expression. 2 S. Mendo| BM24_25 Termination of RNA synthesis (eukaryotic) in Pol II 3 S. Mendo| BM24_25  RNA a central player in gene expression ✓Life may have evolved from an RNA world Ribozyme green rectangles show processes catalyzed by RNA 4 S. Mendo| BM24_25  Types of RNA Non-coding Coding RNA (nc) RNA housekeeping regulatory Circular RNA tRNA and mRNA ncRNAs (circRNA) rRNA Long (l) Small (s) ncRNAs ncRNAs small small- PIWI- micro RNA nucleolar small nuclear interfering interacting (miRNA) RNA RNA (snRNA) RNA (siRNA) RNA (piRNA) (snoRNA) 5 S. Mendo| BM24_25  RNA structures Hydrogen bonding (pairing) between Built hierarchically onto secondary complementary bases structure elements ‣ RNA molecules are negatively charged ‣ The tendency is to repel one another ‣ In solution stable secondary structures are formed ‣ The formation of RNA helices is stimulated by +charged molecules (Mg2+) 6 S. Mendo| BM24_25  RNA processing: intron removal - splicing ✓ Interrupted genes are found in all eukaryotic organisms. ✓Exons are spliced together in a pre-mRNA by the spliceosome ✓Introns mean that most eukaryotic genes are longer than the nal cytoplasmic mRNA ✓ Introns are no junk: they contain regulatory elements and genes ✓ Intron removal is crucial for: i) RNA polymerase transcriptional elongation ii) export of mRNA from the nucleus 7 S. Mendo| BM24_25 fi Alternative splicing occurs in most transcripts of multicellular eukaryotes ✓ Different modes of alternative splicing ✓ Speci c exons or exonic sequences may be excluded or included in the mRNA products by using alternative splicing sites. ✓ Alternative splicing contributes to structural and functional diversity of gene products. We will talk about this in detail later (future classes!) 8 S. Mendo| BM24_25 fi  Types of RNA splicing ✓ Nuclear RNA encoding proteins (cis-splicing): requires conserved sequences in introns and spliceosomes | (GU…AG + branch site/ pyrimidine tract) splicing mechanism: spliceosomal ✓Trans-splicing of nuclear RNA splicing mechanism: spliceosomal ✓Self splicing: ‣ Type/group I and Type /group II splicing mechanism: self splicing ‣ tRNA splicing mechanism: enzymatic ‣ rRNA 9 S. Mendo| BM24_25  Introns enhance eukaryotic gene expression ✓ Can contain cis-acting elements that promote transcription of genes (transcriptional enhancers containing sites for binding of TF, thus increasing transcription by RNA pol II ✓ They can contain genes: protein coding or encoding non-coding RNAs (small nucleolar RNA- snoRNA, miRNA and ncRNAs intron spliced from UGH pre-mRNA 8 snoRNA (U22) ✓ Contribute to timing of gene expression during development ✓ Have an important role in evolution of new proteins 10 S. Mendo| BM24_25  Capping of the mRNA ‣ Shortly after transcription initiation, Pol II is paused 30 nt downstream the ISS +1 waiting for the recruitment of the cap enzymes ✓ Addition of a 5´ terminal G base of the transcript by guanylyl transferase (GT) after transcription; links the terminal P of 5´GTP to the terminal base of mRNA. ✓ The capping process takes place during transcription and may be important for release from pausing of transcription. ✓ cap is substrate for methylation ✓ The cap structure (monomethylated) is recognized by protein factors to in uence mRNA stability, splicing, export, and translation ✓ important for: ‣ protection against 5´- 3´ exonucleases ‣ pol II to enter productive elongation ‣ as checkpoint for transcription to reinitiate 11 S. Mendo| BM24_25 fl  The spliceosome ✓ Large macromolecular complex consisting of several non-coding RNAs and dozens of auxiliary proteins (snRNPs). ✓Frames on pre-mRNA from snRNAs (U1snRNA…..U6) + associated proteins (snRNPs), e.g.: Sm proteins + splicing factors (SF) ✓U1…..U6 (U: high levels of uridine); 1…. 6 abundance in the cell (1- most abundant) ✓Assemble and disassemble on each intron (e.g.: 8 exons; 7 spliceosomes) 12 S. Mendo| BM24_25  The three variant spliceosomes ✓ The major spliceosome: recognizes introns with 5´-GT….AG-3´ and contains U1, U2, U4/U6 and U5 snRNPs ✓The minor spliceosome: recognizes introns with 5´-AT….AC-3´ and contains a different set of snRNPs (except U5) ✓Trans-spliceosome: catalyses the splicing together of separate pre-mRNA molecules which are transcribed from separate genes 13 S. Mendo| BM24_25  Spliceosome can assemble through intron and exon de nition ✓In higher eukaryotes the spliceosome rst recognises the exons within the pre-mRNA- Exon de nition ✓Important for gene expression ✓why is exon de nition important? ‣ size of exons ‣ anked by both 5´ and 3´ splice sites 14 S. Mendo| BM24_25 fl fi fi  What are the rules for intron and exon de nition? ✓Introns with 500 nt: exon de nition (important for alternative splicing and in diseases where pre-mRNA is not properly decoded. ‣ humans and animals: frequent exon de nition ‣ Yeast: intron de nition ‣ anked by both 5´ and 3´ splice sites 15 S. Mendo| BM24_25 fl fi fi fi fi fi  Splicing: a two step chemical reaction ‣ A lariat is formed when the intron is cleaved at the 5′ splice site and the 5′ end is joined to a 2′-OH position at an A at the branch site in the intron. The upstream 3´-OH of the exon is released ‣ The 3´-OH end of the upstream exon attacks the 3´-5´-Pdi ester bond joining the intron and 3´ splice site ‣ The reactions occur by transesteri cations, in which a bond is transferred from one location to another. Can be used as an ncRNA or degraded https://www.youtube.com/watch?v=cyrJEZN3Cbc 16 S. Mendo| BM24_25 fi  Splice sites ✓It is possible to assign a speci c end to every intron (conservation of exon-intron junction) ✓High conservation of nt is found within the intron ‣ Major introns or U2 type follow GU- AG rule (GT-AG DNA sequence) de ned according to the 5´ and 3´ends, respectively ‣ Minor introns or U12 type have AU- AC at its ends They are better classi ed according to the splicing machinery as U2 and U12 introns 17 S. Mendo| BM24_25 fi fi fi  The 3´splice site sequence ✓Comprises 3 distinct parts: Polypirimidin tract ( T´s) 100nt ✓ Splicing depends only on recognition of pairs of splice sites ✓ Additional conserved sequences at both 5′ and 3′ splice sites de ne functional splice sites among numerous other potential sites in the pre-mRNA. ✓ cryptic sites (related sequence used when the branch site is inactivated) can also be used. ✓ The role of the branch site is to identify the nearest splice site. 18 S. Mendo| BM24_25 fi  Intron removal overview: spliceosome assembly https://www.youtube.com/watch?v=ri0SmCf5BnM 19 S. Mendo| BM24_25  Spliceosome assembly and commitment of pre-mRNA to splicing ✓Complementary sequences between snRNAs and conserved splice sites in the pre-mRNA assist on spliceosome assembly and enter in the spliceosome cycle (ILS) intron lariat spliceosome Complex A Pre-catalytical spliceosome Catalytically activated https://doi.org/10.1016/j.sbi.2015.12.005 20 S. Mendo| BM24_25  The precursor messenger RNA (pre-mRNA) splicing cycle degradation of the excised Initially U1 and U2 snRNPs intron lariat recognise the 5′ splice site intron lariat spliceosome (ILS) and the branch point in pre- is disassembled mRNA and recruit the pre- assembled U4/U6. spliced mRNA product second trans- esteri cation reaction Unwinding of the extensively base-paired U4/U6 snRNA duplex by Brr2 helicase recruitment of NTC (nineteen new base-pairing interaction between U2 and U6 complex) and NTR (nineteen snRNAs; position the 2′OH group of he BP to attack related complex) the phosphodiester bond at the 5′ SS https://www.cell.com/cell/fulltext/S0092-8674(19)30155-2 21 S. Mendo| BM24_25 fi  Spliceosome cycle: step 1 ✓ Formation of the H complex ‣ Association of pre-mRNA + hnRNPs (splicing regulators- repressors) ‣ U1 snRNP initiates splicing by binding to the 5′ splice site by means of an RNA–RNA pairing reaction (5´ end remains ss and base pairs with the 5´splice site) U1 snRNP 22 S. Mendo| BM24_25  Spliceosome cycle: step 2 ✓ Formation of the E (early or commitment) complex ‣ Biding of U1 snRNP, splicing factors and regulators (U2AF- U2 auxiliary factor) and SR (a protein with a Ser-Arg rich domain facilitate protein-protein interaction with other splice factors) to the 5´ and 3´ splice sites ‣ Recognition of the splice sites can take two routes: ‣ Introns are long Intron de nition Exon de nition ‣ splice sites are weak Does not require any sequence Sequences downstream outside the intron of the intron are required 23 S. Mendo| BM24_25 fi fi  Spliceosome cycle: step 3 ✓ Formation of the A complex ‣ Displacement of SF (splicing factors) and replacement by U2 24 S. Mendo| BM24_25  Spliceosome cycle: steps 4 & 5 ✓ Formation of the pre-catalytic B complex ‣ Binding of U5 and U4/U6 snRNPs converts the A complex to the B1 spliceosome, which contains all the components necessary for splicing. ‣ The spliceosome passes through a series of further complexes as splicing proceeds. ✓ In the U6/U4 snRNP 26 bp of U6 is paired with two separate regions of U4 ✓ The release of U4 allows (controls) ✓ Release of U1 snRNP allows U6 spliceosome to proceed to the snRNA to interact with the 5′ splice site activated state and converts the B1 spliceosome to the B2 spliceosome. ✓ When U4 dissociates from U6 snRNP, U6 snRNA can pair with U2 snRNA to form the catalytic active site. 25 S. Mendo| BM24_25  Spliceosome cycle: step 6 & 7 ✓ Splicing catalysis: C complex ‣ Contains the three remaining snRNP (U2, U5 and U6) + 100 different proteins ‣ Lacks UsAF and U1 snRNP which are discarded earlier in the cycle. ✓ mRNA is released by the spliceosome ✓ exported to the cytoplasm ✓ the lariat (intron) is degraded ✓ snRNPs are recycled 26 S. Mendo| BM24_25 Summary of spliceosome cycle 27 S. Mendo| BM24_25  Types of RNA splicing ✓ Nuclear RNA encoding proteins (cis-splicing): requires conserved sequences in introns and spliceosomes ✓Trans-splicing of nuclear RNA ✓Self splicing: ‣ Type/group I and Type /group II ‣ tRNA ‣ rRNA 28 S. Mendo| BM24_25 Trans splicing of nuclear RNAs ✓RNA processing event ✓ Creats an RNA from two different transcripts ✓ Occurs in mos eukaryotes (best characterized in C. elegans where 70% of RNAs are trans spliced ✓Different than splicing that only uses one pre-mRNA to make mRNA ✓Can be used for gene therapy techniques ✓Gives rise to transcriptomic diversity (many proteins with few genes) 29 S. Mendo| BM24_25  Trans splicing of nuclear RNAs ✓ Splicing is expected to occur only in cis (sequences of the same RNA are spliced together) ✓ Trans-splicing is rare, but occurs in some organisms: trypanosomes and nematode worms ✓ In trypanosomes trans-splicing of a spliced leader RNA - (SL RNA) is important to prepare mRNA for translation. ✓ SL contains a 39 nt mini exon and a 5´SS; this mini exon RNA donates the 5´ SS for trans- splicing; 100 nt in length; is a snRNP ✓ Transesteri cation reactions occur as in cis-splicing: ‣ 1st: 2´-OH at branch point of an RNA attacks the 5´SS of SL, resulting in a Y- shapped molecule ‣ 2nd: 3´-OH at the end of SL attacks the 3´-OH SS of the exon2 U1 snRNP SS recognition is performed by SL which contains the 5´SS Tr 30 S. Mendo| BM24_25 fi  Trans splicing of nuclear RNAs ‣ It generates chimeric mRNAs https://www.youtube.com/watch?v=cT2fDRivmbA 31 S. Mendo| BM24_25 Trans splicing of nuclear RNAs 32 S. Mendo| BM24_25 Trans splicing of nuclear RNAs 33 S. Mendo| BM24_25  Types of RNA splicing ✓Nuclear RNA encoding proteins (cis-splicing): requires conserved sequences in introns and spliceosomes ✓Trans-splicing of nuclear RNA ✓Self splicing: ‣Type/group I and Type /group II ‣tRNA ‣rRNA 34 S. Mendo| BM24_25  Self-splicing introns (ribozymes) ‣ Group I and Group II (can fold in secondary structures) excise themselves for an mRNA by autosplicing or self-splicing: depends only on the sequence (and 2ary structure) of the intron, and secondary structure of the mRNA, not on proteins!. First identi ed in Tetrahymena (ciliate protozoan)- splicing of the pre-RNA occurred in the absence of any Depends on the Depends on the added protein (free guanosine) free 2´OH G co-factor Linear seq. Lariat https://www.youtube.com/watch?v=lQdBI1xEoaQ 35 S. Mendo| BM24_25 fi  Self-splicing introns ‣ Group I and Group II (can fold in secondary structures) excise themselves from an mRNA by autosplicing or self-splicing: depends only on the sequence of the intron, and secondary structure of the mRNA, not on proteins!. ✓ The splice sites and mechanism of splicing of group II introns are similar to splicing of nuclear introns. ✓ A group II intron folds into a secondary structure that generates a catalytic site resembling the structure of U6-U2- nuclear intron. ✓ 1st a 2’ free -OH attacks the exon1- intro junction, which then attack the intro in exon 2. ✓ Group II introns might have been precursors of modern day pre-mRNA introns 36 S. Mendo| BM24_25 Other variants of introns: U12-dependent introns ✓Minor class of introns following an AT-AC rule (not always!) are found only in metazoans and plants ✓Removed by minor spliceosomes ✓Spliceosome contains U12 snRNP (so better refer to the machinery and not the rule!) ✓Do not have a polypyrimidine tract downstream of the branch point site 37 S. Mendo| BM24_25  tRNAs processing by RNases and splicing ✓tRNA splicing occurs by successive cleavage and ligation reactions, unlike most splicing reactions; it requires 5 enzymes (e.g.: endonuclease, exon ligase, adenylate synthetase, cyclic phosphodiesterase, phosphotransferase); tRNA has 2 exons and 1 intron. ✓Splicing depends on the 2ary structure in tRNA (not on intron sequence) ✓There is no consensus sequence that could be recognised by splicing enzymes. ✓Intron creates an alternative conformation for the anticodon arm. ✓An endonuclease cleaves the tRNA precursors at both ends of the intron. ✓ Pairing between a base in the intron and an nonpaired base in the stem in uence splicing INTRON 38 S. Mendo| BM24_25  tRNAs processing and formation of the anticodon loop Primary transcript Endonuclease Endonuclease Intermediate transcript Base modi cations 5´ cleavage 3´ cleavage CCA addition Release of the intron generates two half-tRNAs with unusual ends that contain 5′ hydroxyl and 2′,3′–cyclic phosphate. Mature transcript Anticodon loop 39 S. Mendo| BM24_25 fi  Production of rRNAs (not really splicing!) ✓ The large and small rRNAs are released by cleavage from a common precursor rRNA (5.8S, 18S and 28S rRNAs) transcribed by pol I (in the nucleolus); the 5S rRNA is separately transcribed by pol III. ✓ Multiple copies are always available ✓rRNA processing takes place cotranscriptionally (processing factors and ribosomal proteins together to build ribosomes) ✓ With the involvement of small nucleolar RNAs (snoRNAs) that appear to help on the formation of a 2ary structure that is recognised by the endonuclease; e.g.:U3 snoRNA is required for the 1st cleavage event ITS2 5´EST ITS1 3´EST Small nucleolar RNAs (snoRNAs) are a class of Endonuclease Endonuclease small RNA molecules that reactions Triming promote modi cations of other RNAs. e.g.: generate mature rRNAs Exonuclease https://www.youtube.com/watch?v=9C31e4IM5Qg 40 S. Mendo| BM24_25 fi rRNAs processing by snoRNAs (not true splicing) No introns are present! 41 S. Mendo| BM24_25  SUMMARY ‣ Different types of intron removal reactions have been identi ed: eukaryotic nuclear introns, Group/type I and II, tRNAs, ‣ Pre-mRNA splicing requires short consensus sequences, whereas the rest of the intron sequence is irrelevant: the majority follow the GU-AG rule; ‣ Pre-mRNA splicing requires formation of a spliceosome, which forms by the process of intron/ exon de nition; pre-mRNA complex contains U1 snRNP and other key proteins including U2AF ‣ The splicesosome contains U1 to U6 snRNP + splicing factors ‣ cis-splicing is quite frequent but trans-splicing occurs in trypanosomes and nematodes, and involves a reaction between an SL RNA and a pre-mRNA ‣ tRNA splicing involves endonuclease and ligase reactions ‣ Hundred of thousands of non-coding RNAs are expressed in eukaryotic cells as snoRNAs 42 S. Mendo| BM24_25 fi fi