Molecular biology - Chapter 16: Other rna processing events

Molecular BiologyFourth EditionChapter 16Other RNA Processing EventsLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.116.1 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 out2Eukaryotic 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 another3Human Cells Processing5’-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-pairing4Bacterial 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 precursor5Processing Bacterial rRNA616.2 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, precursor may contain one or more tRNA7Cutting 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 III8Forming 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 subunit9RNase 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’-ends10Forming 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 nucleotide11Processing 3’-Ends1216.3 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 SL to the Coding Region of an mRNA15Trans-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 intermediate1616.4 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 RNAs17Mechanism 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’-ends18Role 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 region19RNA Editing20Guide RNA Editing5’-end of the first gRNA hybridizes to an unedited region at the 3’-border of editing I 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 21Mechanism 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 TUTase22Editing 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 properly2316.5 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 geneIt is caused by an increase in half-life of casein mRNA24Transferrin 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)25Rapid 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 bind26Determining TfR mRNA StabilityWhen iron concentration is high, TfR mRNA decays rapidlyWhen iron concentration is low, TfR mRNA decays much more slowly Difference is about 20-foldImportant in determining the rate of accumulation of TfR mRNATRS-3 mRNA Has no rapid turnover determinantIs constitutively stableTRS-4 mRNA has no ability to bind IRE-binding protein and is constitutively unstable27TfR mRNA Degradation PathwayInitiating 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 stability28Role of Iron LevelWhen iron concentration is low, aconitase exists in an apoprotein form lacking ironBinds to the IREs in the TfR mRNA and protects RNA against attack by RNaseHigh iron concentration Aconitase apoprotein binds to ironCannot bind to TfR mRNA IREsLeaves RNA vulnerable to degradation29Destabilization of TfR mRNA by Iron30RNA InterferenceRNA interference occurs when a cell encounters dsRNA from:VirusTransposonTransgene Trigger dsRNA is degraded into 21-23 nt fragments (siRNAs) by an RNase III-like enzyme called DicerDouble-stranded siRNA, with Dicer and Dicer-associated protein R2D2 form a complex called complex B31Complex BComplex B delivers the siRNA to the RISC loading complex (RLC)Separates 2 strands of siRNATransfers guide strand to RNA-induced silencing complex (RISC) that introduces a protein, Ago2The 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 degradation32Amplification of siRNAsiRNA is amplified during RNAi when antisense siRNAs hybridize to target mRNA and prime synthesis of full-length antisense RNA by RNA-dependent RNA polymeraseNew dsRNA is digested by Dicer into new pieces of siRNA33Role of RNAi Machinery in Heterochromatin FormationRNAi machinery is involved in heterochromatization at yeast centromeres and silent mating-type regionsIn fission, yeast at the outermost regions of centromeres active transcription of the reverse strand occursForward transcripts can base-pair with the reverse transcript to kick off RNAiIn turn this recruits a histone methyltransferase that methylates Lys-9 of H3This recruits Swi6 causing heterochromatization34Heterochromatin Formation in Plants and MammalsIn plants and mammals, formation of heterochromatin is abetted by DNA methylationThis methylation can also attract heterochromatization machineryIndividual genes can be silenced in mammals by RNAi that targets the gene’s control region rather than the coding regionSilencing process involves DNA methylation rather than mRNA destruction 35MicroRNAs and Gene SilencingMicroRNAs are 18-25 nt RNAs produced naturally in plant and animal cells by cleavage from 75-nt stem-loop precursor RNAIn last step of miRNA synthesis, Dicer RNase cleaves ds stem part of the precursor to yield miRNA in ds formSingle-stranded form of miRNAs can team up with Argonaute protein in a RISC to control gene expression by base-pairing to their mRNAs36Pathways to Gene Silencing by miRNAs37Animal and Plant miRNAIn animals, miRNAs tend to base-pair imperfectly to the 3’-UTRs of their target mRNAsThis leads to inhibition of protein protein product accumulation of such mRNAIn plants, miRNAs tend to base-pair perfectly or near-perfectly with target mRNAsThere are some exceptions which can lead to translation blockage38