UC Biologia Molecular Past Paper 2024-2025 - Part 2, Module 4

UC Biologia Molecular| Ano lectivo 24_25 Parte II| Aula: 4 3 e 4 dezembro Sumário: Transporte nucleo-citoplamático do mRNA: estabilidade e localização do mRNA. Transporte do mRNA para o citoplasma. Estabilidade dos mRNAs. Vias de degradação dos mRNAs eucarióticos depe...

UC Biologia Molecular| Ano lectivo 24_25 Parte II| Aula: 4 3 e 4 dezembro Sumário: Transporte nucleo-citoplamático do mRNA: estabilidade e localização do mRNA. Transporte do mRNA para o citoplasma. Estabilidade dos mRNAs. Vias de degradação dos mRNAs eucarióticos dependentes de deadenilação. O tempo de meia-vida dos mRNAs são controladas por diferentes sequências ou estruturas. Os mRNAs passam por um processo de veri cação de controlo de qualidade. Localização de mRNAs eucarióticos. Nucleo-cytoplasmic transport of mRNA: mRNA stability and localization. Transport of mRNA to the cytoplasm. Stability of mRNAs. Deadenylation-dependent degradation pathways of eukaryotic mRNAs. The half-lives of mRNAs are controlled by different sequences or structures. The mRNAs go through a quality control veri cation process. Localization of eukaryotic mRNAs. 1 S. Mendo| BM24_25 fi fi  Di erent mechanisms of export of mRNA and nc RNAs ✓Export challenges were solved very early in eukaryotic evolution Mex67, Mtr2 and Arx1: auxiliary exporters (yeast only) general exporter ‣ export adaptors (blue); export receptors (yellow); additional adaptor proteins and RNA binding factors (orange ovals) 2 S. Mendo| BM24_25 ff  mRNA export from the nucleus ‣ Export through the NPC requires export adaptors and receptors as well as Dbp5p (helicase) Once in the cytoplasm, some Other mRNPs are transported to mRNPs are stored in a speci c subcellular translationally silent state locations along the 5 major steps: cytoskeleton 1. mRNA is coated with nuclear proteins to give rise to export competent mRNP (mRNA+ proteins) 2. mRNP associates with nuclear export receptor (NER) 3. mRNA passes through nuclear pores 4. Disassembly of export competent mRNP 5. Re-import of shuttling proteins into nucleus for next round of exports Eukaryotic translation initiation factor 4F (eIF4F) is a protein complex that mediates recruitment of ribosomes to mRNA 3 S. Mendo| BM24_25 fi  Proteins involved in nuclear export of mRNA ✓ Export adaptors: TREX (transcription export) complex and SR proteins (rich in ser/ arg)| link mRNA with transport apparatus protein complex involved in elongation RNA export factor REF THO and mRNA Binds to nuclei export export receptor UAP56 RNA helicase Component of the spliceosome ✓3´end formation: Polyadenylation proteins| release transcript from the gene ✓Export receptors: TAP/ NXT/F (metazoans)/ Mex67 (yeast)| bind to both export adaptors and nuclear pore ( REF: RNA export factor- export adaptor that has a motif that binds to RNA) ✓Nuclear pore proteins: FG repeat nucleoporins| found in the inner channel of the nuclear pore interface interacting with RNA export complex as it leaves the nucleus FG Nups-(phenylalanine-glycine repeats) that bind karyopherins and facilitate transport of karyopherin-cargo complexes ✓Disassembly and recycling of export components: DBP5 RNA helicases| disassemble RNA export complexes once they reach cytoplasm 4 S. Mendo| BM24_25 The TREX complex ✓ Full assembly of TREX complex signals that the mRNA processing is complete and mRNA is ready to export ✓It is stabilised on the mRNA by protein interactions with cap binding complex (CBC) ✓REF only binds to mRNA strongly when it interacts with UAP56 in the TREX complex ✓THO complex contains several proteins; Tho2 is involved in transcription elongation and mRNA export. 5 S. Mendo| BM24_25 Nuclear export of mRNA facilitated by TREX complex https://doi.org/10.1016%2Fj.bbagrm.2011.12.001 NFX proteins have been implicated in the export of mRNAs from the nucleus 6 S. Mendo| BM24_25 The role of SR proteins as RNA export adaptors TAP TAP ✓SR proteins require phosphorylation to mediate spliceosomal complex formation ✓dephosphorylation is required for splicing catalysis to occur, as well as nuclear export ✓re-entry into the nucleus requires rephosphorylation ✓hyperphosphorylation of SR proteins represses splicing ✓ Hypophosporylated SR remains bound to mRNP and are able to interact with TAP and the mRNA is exported 7 S. Mendo| BM24_25  Contribution of splicing to nuclear export of mRNA ✓Splicing physically adds both SR and TREX onto mRNAs ✓Splicing modi es the SR protein mRNA export adaptor to remove P groups ✓Splicing removes introns from transcripts: introns act as signals to retain RNAs within the nucleus Thus splicing has to be nished before mRNA can be exported How about histones? (mRNAs are not spliced) ✓They still use splicing related proteins (SR) in their export. How about intronless genes? ✓E.g.: S. cerevisiae: TREX binds as part of RNA pol II 8 S. Mendo| BM24_25 fi fi  Polyadenylation is required for mRNA export mRNA 3´end made by 5´ AAAn 3´ GFP Exported normal Poly(A) site mRNA 3´end made by 5´ AAAn 3´ GFP RZ Not Exported ribozyme mRNA 3´end made by ribozyme with upstream 5´ GFP AAAn RZ AAAn 3´ Exported poly(A) site 9 S. Mendo| BM24_25  Addition of nuclear export receptors ✓Transport across the nuclear pore requires nuclear export receptors (TAP and p15) which bind to nuclear adaptors ✓Role of TAP and p15: move mRNAs through nuclear pore ✓It was shown that mRNA accumulates on the nucleus when levels of TAP and p15 (mex67 the homologous in yeast) are low. ✓ TAP can not bind mRNA (it bonds though adaptors) therefore TAP does not exports mRNA that lack adaptors (not ready for export) 10 S. Mendo| BM24_25  Movement of mRNP through the nuclear pore ✓ mRNP go through the nuclear pores which are made up of proteins (nucleoporins) which form a nuclear pore complex (NPC) ✓ 1000 to 10000 nuclear pores in a vertebrate somatic cell, performing ~1000 translocations/s; ✓mRNA doesn’t not move freely, it passes through a mesh of hydrophobic FG (phenyl alanine; glycine) repeat nucleoporins; ✓ FG repeats form a brush- like structure which acts as a sieve - binding sites for proteins involved in nuclear export: block export of large molecules from the nucleus, allowing passage of small molecules. ✓ allow the passage of proteins and their cargo into the cytoplasm 11 S. Mendo| BM24_25  Disassembly of the export competent mRNP ✓ Movement of mRNP is unidirectional, that is to say it can not return to the nucleus ✓ DBP5 (RNA helicase) ensures that backsliding does not occur by removing the nuclear export receptor TAP from the mRNP once it reaches the cytoplasm (mRNP can not reenter the hydrophobic environment of the nuclear pore. ✓DBP5, joins the mRNA as it is being transcribed, but it is only activated to remove TAP, once in the cytoplasm. 12 S. Mendo| BM24_25  Shuttling of export components back to the nucleus ✓ mRNA export adaptors are shuttled back into the nucleus so that they won’t accumulate in the cytoplasm and allow further mRNA export. 13 S. Mendo| BM24_25  The mRNA encoded information ✓ Synthesis of a speci c polypeptide ‣ How frequently it will be translated? ‣ How long it will survive (half-life)? ‣ Where it will be translated? Information carried out in cis-elements and proteins non-protein coding regulatory information 14 S. Mendo| BM24_25 fi  mRNAs are unstable ✓ Weaker phosphodiester bonds (2´OH) ✓ Action of ribonucleases. ✓Ribonucleases differ in their substrate preference and mode of attack. ‣ participate in DNA replication ‣ DNA repair ‣ processing of new transcripts ‣ degradation of mRNA (Endoribonuclease (endonuclease) – cleaves an RNA at internal site(s); Exoribonuclease (exonuclease) – removes terminal ribonucleotides from RNA). 15 S. Mendo| BM24_25  mRNAs half-life ✓ mRNA stability is encoded in cis-sequences and is characteristic of each mRNA ✓ Half- life varies ‣ E. coli: ~3 minutes ( 20 seconds - 90 minutes) ‣ Yeast: 3-100 minutes ‣ Metazoans: minutes, to hours or even days! ✓mRNA decay – mRNA degradation, assuming that the degradation process is stochastic. 16 S. Mendo| BM24_25 Prokaryotic mRNA degradation ✓Degradation of bacterial mRNAs is initiated by removal of a pyrophosphate from the 5′ terminus. ✓Monophosphorylated mRNAs are degraded during translation in a two-step cycle involving endonucleolytic cleavages, followed by 3′–5′ digestion of the resulting fragments. ✓This process proceeds very rapidly ✓The main degradation enzymes work as a complex called the degradosome (RNAse E+ PNPase- endonucleases + helicase + proteins). 17 S. Mendo| BM24_25  Eukaryotic mRNA degradation: ✓The two major mRNA decay pathways are initiated by deadenylation catalyzed by poly(A) nucleases. ✓Cap is resistant to decapping during translation ✓Poly (A) tail gradually shortens upon entry into cytoplasm (by deadenylases). 1) Deadenylation may be followed either by decapping and 5′–3′ exonuclease (xrn1) digestion or by 3′–5′ exonuclease digestion (by Dcp1 and 2). 2) The exosome, a large conserved complex, catalyzes 3′–5′ mRNA digestion. 1) 5´to 3`decay 3´to 5`decay 2) Dcp1 and Dcp2: decapping enzymes 18 S. Mendo| BM24_25  Other degradation pathways target speci c mRNAs Protein S28 recruits a decapping enhancer. Decapping is autoregulated: more S28, more accelerated mRNA decay Histones mRNA are degraded after S-phase. Similar to bacteria: addition of a PolyU tail, which acts as platform for exosome and /or LSM1-7, activating decay Cleavage followed by directionally digestion of fragments; a decapping enzyme removes cap. Directs endonucleolytic cleavage of mRNA in plants; translation repression in animals (1000 mi RNAs in humans) RNA induced silencing complex b) The degradation of the non polyadenylated histone mRNAs is initiated by 3′ addition of a poly(U) tail. c) Degradation of some mRNAs may be initiated by sequence-speci c or structure- speci c endonucleolytic cleavage. d) An unknown number of mRNAs are target for degradation or translational repression by microRNAs. 19 S. Mendo| BM24_25 fi fi mRNA-speci c half-lives are controlled by sequences or structures Speci c cis-elements ( within 3´UTR) in an mRNA affect its rate of degradation (mRNA half-life). ✓ Destabilizing elements (DEs) can accelerate mRNA decay, while stabilizing elements (SEs) can reduce it (they have stabilizing Py rich sequences in the 3´UTR). ✓ AU-rich elements (AREs) are common destabilizing elements in mammals and are bound by a variety of proteins. They recruit degradation machinery. E.g.: AUUUA; U-rich (appear once or repeated in the genome) ✓ Some DE-binding proteins interact with components of the decay machinery and probably recruit them for degradation. 20 S. Mendo| BM24_25 fi fi mRNA-speci c half-lives are controlled by sequences or structures ✓SEs occur on some highly stable mRNAs; mRNA degradation rates can be altered in response to a variety of signals. ✓ARE-BP prevent ARE from destabilising mRNA E.g.: mammalian transferrin mRNA (responsible for ferric-ion delivery to tissues) Iron response element : more transfers needed to import Fe from blood stream 3´UTR 21 S. Mendo| BM24_25 fi  RNA-checking for defects ✓Many improperly processed transcripts are not exported from the nucleus, are restricted to the site of transcription, and are in some cases degraded. 22 S. Mendo| BM24_25  RNA-checking for defects ✓ Errors are made at all steps of processing ✓Aberrant nuclear RNAs are identi ed and destroyed by an RNA surveillance system (exist in the nucleus and in the cytoplasm). ‣ Identify and tag the aberrant/misfolded RNA ‣ Destroy it (exosome) ✓The nuclear exosome functions both in the processing of normal substrate RNAs and in the destruction of aberrant RNAs. ✓The TRAMP complex targets aberrant RNAs to the exosome and facilitates its 3′–5′ exonuclease activity. 1) interacts with exosome, stimulating exonuclease activity 2) Includes an RNA helicase: unwinds 2ary structure, removes RBPs 3) A non-canonical poly (A) polymerase which adds an oligo (A) tail to the target substrate 23 S. Mendo| BM24_25 fi  Quality control of mRNA translation delity ✓ Surveillance systems detect 3 types of RNA defects and target them to degradation: 1) Non-sense termination decay: ‣ targets mRNA containing a premature termination codon (PTC)- nonsense mutation; ‣ It produces C- truncated polypeptides TC: termination codon considered toxic to the cell; ‣ found in all eukaryotes. ‣ Targeting requires Upf proteins ‣ important rapid decay pathway for short lived mRNAs How are PTCs distinguished from normal termination codon? ‣ Presence of a splice junction; involves unusual 3′ UTR structure and the presence of downstream exon junction complexes (EJCs) downstream of PTC, in mammals. ‣ Presence of a too long 3´UTR 24 S. Mendo| BM24_25 fi  Nonsense termination decay ‣ In probably all eukaryotes an abnormal long 3´UTR is recognised by the distance between the TC and the poly (A)-PABP complex 25 S. Mendo| BM24_25  Quality control of mRNA translation delity ‣ Targets mRNA that lack an in-frame termination 2) Nonstop decay: codon ‣ Ribosome translates into poly(A) tail ‣ Premature transcription termination ‣ Requires a conserved set of proteins SKI ‣ SKI7 binds to ribosome A site to stimulate release ‣ NSD prevents accumulation of toxic polypeptides and liberation of trapped ribosomes ‣ Targets mRNA with ribosome stalled at coding 3) No-go decay: region - strong secondary structures - rarely used codons (cognate tRNAs are not available) ‣ Is the least understood ‣ mRNA degradation is initiated by an endonucleolytic cut and the 5´-3´ fragment is digested by Xrn1 26 S. Mendo| BM24_25 fi The mechanisms of localization, translation and degradation are interconnected Generally LOCALIZATION Don’t translate until localised TRANSLATION Stop translation before degradation DEGRADATION 27 S. Mendo| BM24_25  RNA repression during transport inhibition of + binding of subunits 40S and 60 S ‣ Puf6p and Khd1p block AHS1 mRNA translation during transport 28 S. Mendo| BM24_25  mRNA localization ‣ Some mRNAs are translated at speci c sites, though translation is repressed until they reach their destination ‣ Localization requires cis-elements on the target mRNA and trans-factors to mediate localization In oocytes (germinal granules): set up patterns in the embryo and to assign developmental fates to cells in different regions (maternal RNAs are translated in speci c times and places of the development of the embryo) In cells that divide assimetrically, is a mechanism to segregate protein factors to only one of the daughter cells In polarized cell types, it is a mechanism to establish cellular compartments 29 S. Mendo| BM24_25 fi fi  mRNA localization ‣ In some cases mRNA localization involves transport from one cell to another ‣ Three general mechanisms are known - mRNA is uniformly distributed but degraded except at the site of translation - mRNA is initially freely diffusible, but it becomes trapped at the site of translation - mRNA is actively transported to the site where it is translated; this is the predominant mechanism for localization achieved by translocation of motor proteins (dyneins, kinesins and myosins) along cytoskeletal tracks 30 S. Mendo| BM24_25 The active transport mode of localization ‣ Requires four components: - cis-elements (zip codes: localization signals) on the target mRNA - 3´UTR - trans-factors that directly or indirectly attach the mRNA to the correct motor protein - trans-factors that repress translation or mediate degradation of unlocalized mRNAs - an anchoring system at the desired location 31 S. Mendo| BM24_25  Loss of localization may underlie pathology ‣ E. g.: In mammals FMRP (fragile X mental retardation protein) directs mRNA to neurons; loss of proper localization leads to the pathology ‣ Fragile X syndrome is caused by the absence of functional fragile X mental retardation protein (FMRP), an RNA binding protein ‣ FMRP regulates the local translation of a subset of mRNAs at synapses in response to activation of Gp1 metabotropic glutamate receptors (mGluRs) and possibly other receptors ‣ FMRP plays a role in regulated mRNA transport in dendrites 32 S. Mendo| BM24_25  Localization mechanism in the budding yeast ‣ During budding ASH1 mRNA is localized to the developing tip; thus, Ash1 protein synthesis occurs only at the daughter cells. ‣ Newly exported ASH1 mRNA is attached to the myosin motor Myo4 via a complex with the She2 and She3 proteins. The motor transports the mRNA along actin laments to the developing bud. 33 S. Mendo| BM24_25 fi mRNA cycle and regulation 34 S. Mendo| BM24_25  SUMMARY ‣ Export of mRNA involves 5 different classes of proteins which bind sequentially to the mRNA leading to its export from the nucleus - nuclear-cytoplasmic take place through NPC - NPC is made up of nucleoporins - RNAs are exported complexed with proteins - TAP is the RNA export receptor, which are linked to export adaptors - mRNP associate with FG, transversing the hidrophobic moiety of the NP. ‣ mRNAs are unstable and are degraded by multi enzymes ‣ Most eukaryotic mRNAs are degraded via two deadenylation-dependent pathways ‣ mRNA half-lives are controlled by sequences or structures ‣ mRNAs are checked for quality control ‣ Some eukaryotic mRNAs are localised to speci c regions in the cell. 35 S. Mendo| BM24_25 fi