Bài giảng Molecular Biology - Chapter 16 Other Post-Transcriptional Events

Molecular BiologyFifth EditionChapter 16Other Post-Transcriptional EventsLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.1Other Processing EventsIn a few organisms, other specialized pre-mRNA processing events occur, such as trans-splicingMost organisms process their rRNAs and tRNAs by more conventional mechanismsEukaryotes control some of their gene expression by regulating posttranscriptional processes, primarily mRNA degradation216.1 Ribosomal RNA ProcessingrRNA genes of both eukaryotes and bacteria are transcribed as larger precursors must be processed to yield rRNAs of mature sizeSeveral different rRNA molecules are embedded in a long, precursor and each must be cut out3Eukaryotic rRNA ProcessingRibosomal RNAs are made in eukaryotic nucleoli as precursors that must be processed to release mature rRNAsOrder of RNAs in the precursor is 18S5.8S28S in all eukaryotesExact sizes of the mature rRNAs vary from one species to another4Processing scheme of human rRNA precursor5’-end of 45S precursor RNA is removed to 41S41S precursor is cut into 2 parts:20S precursor of 18S32S precursor of 5.8S and 28S rRNA3’-end of 20S precursor removed, yielding mature 18S rRNA32S precursor is cut to liberate 5.8S and 28S rRNA5.8S and 28S rRNA associate by base-pairing5Bacterial rRNA ProcessingBacterial rRNA precursors contain tRNA and all 3 rRNArRNA are released from their precursors by RNase III and RNase ERNase III is the enzyme that performs at least the initial cleavages that separate the individual large rRNAsRNase E is another ribonuclease that is responsible for removing the 5S rRNA from the precursor6Processing bacterial rRNA precursors716.2 Transfer RNA ProcessingTransfer RNAs are made in all cells as overly long precursorsThese must be processed by removing RNA at both endsNuclei of eukaryotes contain precursors of a single tRNAIn bacteria, a precursor may contain one or more tRNA8Cutting Apart Polycistronic PrecursorsIn processing bacterial RNA that contain more than one tRNAFirst step is to cut precursor up into fragments with just one tRNA eachCutting between tRNAs in precursors having 2 or more tRNACutting between tRNAs and rRNAs in precursorsEnzyme that performs both chores is the RNase III9Forming Mature 5’-EndsExtra nucleotides are removed from the 5’-ends of pre-tRNA in one step by an endonucleolytic cleavage catalyzed by RNase PRNase P from bacteria and eukaryotic nuclei have a catalytic RNA subunit called M1 RNASpinach chloroplast RNase P appears to lack an RNA subunit10RNase P ActionRNase P makes a cut at the site that becomes mature 5’-end of a tRNAThis enzyme is all that is needed to form mature 5’-ends11Forming Mature 3’-EndsRNase II and polynucleotide phosphorylase cooperate To remove most of extra nucleotides at the end of a tRNA precursorStopping at the +2 stage, with 2 extra nucleotides remainingRNases PH and T are most active in removing the last 2 nucleotides from RNARNase T is the major participant in removing very last nucleotide1216.3 Trans-SplicingSplicing that occurs in all eukaryotic species is called cis-splicing because it involves 2 or more exons that exist together in the same geneAlternatively, trans-splicing has exons that are not part of the same gene at all, may not even be on the same chromosome13The Mechanism of Trans-SplicingTrans-splicing occurs in several organismsParasitic and free-living wormsFirst discovered in trypanosomesTrypanosome mRNA are formed by trans-splicing between a short leader exon and any one of many independent coding exons14Joining the Spliced Leader (SL) to the Coding Region of an mRNA(a)(b)15Trans-Splicing SchemeBranchpoint adenosine within the half-intron attached to the coding exon attacks the junction between the leader exon and its half-intronCreates a Y-shaped intron-exon intermediate analogous to the lariat intermediate16Trans-SplicingTrans-splicing is very widespread in some organismsIn C.elegans all or nearly all mRNAs are trans-spliced to a small group of spliced leadersSuch a group of genes resembles a prokaryotic operon in that they belong to a transcription unit controlled by a single promoterIt differes from a true operon in that the primary transcript is ultimately broken into pieces by trans-splicing, with each coding region being supplied with its own leader1716.4 RNA EditingPseudogenes are a duplicate copy of a gene that has been mutated so it does not function and is no longer usedCryptogenes are incomplete genesTrypanosomatid mitochondria encode incomplete mRNA that must be edited before being translatedEditing occurs in the 3’5’ direction by successive action of one or more guide RNAs18Mechanism of EditingUnedited transcripts can be found along with edited versions of the same mRNAsEditing occurs in the poly(A) tails of mRNAs that are added posttranscriptionallyPartially edited transcripts have been isolated, always edited at their 3’-ends but not at their 5’-ends19Model for the role of gRNA in EditingGuide RNAs (gRNA) could direct the insertion and deletion of UMPs over a stretch of nucleotides in the mRNAWhen editing is done, gRNA could hybridize near the 5’-end of newly edited region20RNA Editing21Guide RNA Editing5’-end of the first gRNA hybridizes to an unedited region at the 3’-border of editing in the pre-mRNAThe 5’-ends of the rest of the gRNAs hybridize to edited regions progressively closer to the 5’-end of the region to be edited in the pre-mRNAAll of these gRNAs provide A’s and G’s as templates for the incorporation of U’s missing from the mRNA 22Mechanism of Removing U’sSometimes the gRNA is missing an A or G to pair with a U in the mRNAIn this case the U is removedMechanism of removing U’s involvesCutting pre-mRNA just beyond U to be removedRemoval of U by exonucleaseLigating the two pieces of pre-mRNA togetherMechanism of adding U’s uses same first and last stepMiddle step involves addition of one or more U’s from UTP by TUTase23Editing by Nucleotide DeaminationSome adenosines in mRNAs of higher eukaryotes, including fruit flies and mammals, must be deaminated to inosine posttranscriptionally for mRNA to code for proper proteinsEnzymes know as adenosine deaminases active on RNAs (ADARs) carry out this kind of RNA editingSome cytidines must be deaminated to uridine for an mRNA to code properly2416.5 Posttranscriptional Control of Gene ExpressionA common form of posttranscriptional control of gene expression is control of mRNA stabilityWhen mammary gland tissue is stimulated by prolactin, synthesis of casein protein increases dramaticallyMost of this increase in casein is not due to increased rate of transcription of the casein gene - it is caused by an increase in half-life of casein mRNA25Transferrin Receptor mRNA StabilityTransferrin receptor-TfR concentration is low when iron concentration is highLoss of TfR is largely due to decreased stability of the TfR mRNAResponse to iron depends on the 3’-UTR of the mRNA which contains 5 stem loops called iron response elements (IREs)26Rapid Turnover DeterminantInstability of this mRNA is caused by a rapid turnover determinant that lies in the 3’-UTRIron response elements A and E along with the large central loop of the TfR 3’-UTR can be deleted without altering the response to ironRemoving all of the IREs, or either 1 or 2 non-IRE stem loops renders the TfR mRNA constitutively stableEach of the non-IRE stem loops and one of IREs B to D are part of a rapid turnover determinantRemoving a C from IREs B-D render the TfR mRNA constitutively unstable, unable to bind27TfR mRNA StabilityInitiating event in TfR mRNA degradation seems to be endonucleolytic cleavage of mRNA more than 1000 nt from its 3’-end within the IRE regionCleavage does not require prior deadenylation of mRNAIron controls TfR mRNA stability28Destabilization of TfR mRNA by Iron29RNA InterferenceRNA interference occurs when a cell encounters dsRNA from a virus, transposon or transgene Trigger dsRNA is degraded into 21-23 nt fragments (siRNAs) by an RNase III-like enzyme called DicerThe guide strand of siRNA base-pairs with target mRNA in the active site of PIWI domain of Ago2Ago2 is an RNase H-like enzyme known as a slicerSlicer cleaves the target mRNA in middle of the region of its base-pairing with the siRNAATP-dependent step has cleaved RNA ejected from RISC which then accepts a new molecule of mRNA for degradation30A simplified model for RNAi31Amplification of siRNAsiRNA is amplified during RNAi when antisense siRNAs hybridize to target mRNA and prime synthesis of full-length antisense RNA by RNA-directed RNA polymerase (RdRP)New dsRNA is digested by Dicer into new pieces of siRNA32Amplification of siRNA33Piwi-interacting RNAs and transposon controlDNA elements known as transposons can transpose or jump place to place in a genomeIn doing so they can interrupt and inactivate genes or even break chromosomesIt is important that cells are able to control transposition, particularly in germ cellsGerm cells produce a class of small RNAs called Piwi-interacting RNAs (piRNAs)Animal somatic cells do not produce piRNAs but produce endogenous siRNAs complementary to transposon mRNAs34Control of TranspositionTransposition of transposons is blocked in animal germ cells by a ping-pong amplification and mRNA destruction involving piRNAsA piRNA complementary to a transposon mRNA binds to Piwi or Aubergine and then base-pairs to a transposon mRNA which initiates cleavage of the transposon mRNA by a slicer activity of the Piwi protein, and the 3’ end of the transposon mRNA is also processedThis generates a mature piRNA that can participate in a new round of transposon mRNA destruction and piRNA amplification35RNAi and HeterochromatizationIs RNAi responsible for histone methylation, and the resulting heterochromatization of the centromere?Various experimental data suggested that RNAi is involved in heterochromatin silencing at the centromereMartienssen and colleagues proposed that the abundant reverse transcripts of the otr region base-pair with forward transcripts produced occasionally by RNA polymerase, or perhaps by RdRP, to form trigger dsRNA36RNAi and HeterochromatizationDicer then digests this dsRNA to produce siRNA, and the siRNA associates with an Argonaute1 protein (Ago1) in a complex called RITSRITS can then attract RdRP in a complex known as RDRC, which amplifies the double-stranded siRNARITS then causes the recruitment of a histone H3 lysine 9 methyltransferaseMethylated lysine 9 recruits Swi6, which is required for forming heterochromatin37RNAi and Heterochromatization38Silencing MechanismsIndividual genes can be silenced in mammals by RNAi that targets the gene’s control region, a promoter-associated transcript) rather than the coding regionAnother silencing mechanism targets nuclear RNA as endogenous double-stranded siRNAs that contain a nuclear localization signal can enter the nucleus and cause degradation of nuclear RNAs by the RNAi mechanismNote that the nuclear location distinguishes this mechanism from ordinary RNAi, which occurs in the cytoplasm39Silencing MechanismsYet another silencing process involves DNA and histone methylation rather than mRNA destruction Silencing due to methylation is considered to be an epigenetic modification as it is not a true genetic change to the sequence of the DNAAlthough it is every bit as important as a genetic change because it can cause the silencing of a gene or even heterochromatization of a whole region of a chromosome40Transcriptional Gene Silencing in PlantsFlowering plants have two nuclear RNA polymerases, pol IV and V, that are not found in animals and fungiPol IV makes siRNAs corresponding to chromatin regions to be silencedPol V makes longer RNAs from regions throughout the plant genome that attract siRNA-Ago4 complexes, but only to regions that are targets for silencing, from which these siRNAs were madeThese complexes in turn attract the enaymes required to methylate both DNA and histones, which leads to heterochromatization41MicroRNAsAnother class of small RNAs called microRNAs (miRNAs) participate in gene silencing They are 22-nt RNAs produced naturally in plant and animal cells by cleavage from larger, stem-loop precursor RNAIn animals, these miRNAs base-pair with the 3’-UTR of specific mRNAs and silence gene expression by blocking translationIn plants, these miRNAs base-pair with the interior of mRNAs and direct the cleavage of those mRNAs42Pathways to Gene Silencing by miRNAs43miRNAs and Antiviral ActivityMicroRNAs do not serve solely as modulators of cellular gene activityEvidence suggests that they also act as antiviral agents by targeting viral mRNAsDavid and colleagues demonstrated in 2008 that interferon- stimulates the production of many miRNAs and that several were complementary to parts of the hepatitis C virus44Major Hypotheses for miRNA Action1 - Blocking translation initiation2 - Blocking translation elongation3 - Degradation of mRNAsHow do we reconcile all these ideas?To date there is clear evidence for multiple mechanisms even within the same organismIs it possible that different miRNAs act in different ways? Or that the same miRNA can act in different ways depending on the cellular context?More studies are needed to fully answer these questions45Stimulation of Translation by miRNAsMicroRNAs can activate, as well as repress translationSteitz and colleagues discovered positive action by miRNAs - they found that the ARE of the human TNF mRNA activates translation during serum starvation, which arrests the cell cycle in the G1 phaseIn particular, miR369-3, with the help of AGO2 and FXR1, activate translation of the TNF mRNA in serum-starved cellsmiR369-3, with the help of AGO2, can repress translation of the mRNA in synchronized cells growing in serum46Biogenesis of miRNAsRNA polymerase II transcribes the miRNA precursor genes to produce primary-RNA (pri-RNA), which may encode more than one miRNAProcessing a pri-miRNA to a mature miRNA is a two-step process1 - a nuclear Rnase III (Drosha) cleaves the pri-miRNA to release a 60-70nt stem-loop RNA, now called the pre-miRNA2 - In the cytoplasm dicer cuts the pre-miRNA within the stem to release a mature ds miRNA47Translation Repression, mRNA Degradation and P-BodiesProcessing bodies (P-bodies or PBs) are discrete collections of RNAs and proteins that are involved in mRNA decay and translational repressionThese cellular foci are enriched in enzymes that deadenylate mRNAs, decap mRNAs and catalyze 5’ to 3’ degradation of mRNAsDegradation of mRNA occurs by a non-RNAi-like mechanism 48Translation Repression, mRNA Degradation and P-BodiesP-bodies are cellular foci where mRNAs are destroyed or translationally repressedGW182 is an essential part of the Drosophila miRNA silencing mechanism in P-bodiesAGO1 probably recruits GW182 to an mRNA within a P-body that marks the mRNA for silencingGW182 and AGO1-mediated RNA decay in P-bodies appears to involve both deadenylation and decapping followed by mRNA degradation by a 5’-3’ exonuclease49Relief of Repression in P-BodiesAlthough many mRNAs are degraded in P-bodies, many others are held and repressed thereIn a liver cell line (Huh7), translation of the CAT-1mRNA is repressed by the miRNA miR-122 and the mRNA is sequestered in P-bodiesUpon starvation, the translation repression of the CAT-1 mRNA is relieved and the miRNA migrates from P-bodies to polysomesThis derepression and translocation of the mRNA depends on the mRNA-binding protein HuR and on its binding site in the 3’-UTR of the mRNA50Other Small RNAsSince the discoveries of siRNAs, miRNAs and piRNAs, other small RNAs have been found, although the functions of these RNAs are largely still unknownOne example is the endo-siRNAs of Drosophila, which are made from Drosophila genes as dsRNA precursorsThese RNAs blur the line between siRNAs and miRNAsThey may protect somatic cells from transposons51