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RNA Processing and Editing Ribosomal RNA Processing – rRNA genes of both eukaryotes and bacteria are transcribed as larger precursors and must be processed to yield rRNAs of mature size – Several different rRNA molecules are embedded in a long, precursor and each must be cut out Ribosomal RNA Processing rRNA genes are organized in clusters separated by nontranscribed spacers (NTS) Eukaryotic rRNA Processing • Ribosomal RNAs are made in eukaryotic nucleoli as precursors that must be processed to release mature rRNAs • Order of RNAs in the precursor is – 18S – 5.8S – 28S in all eukaryotes – Exact sizes of the mature rRNAs vary from one species to another • rRNA processing requires complex of small nucleolar RNAs (snoRNAs) and small nucleolar ribonucleoproteins (snoRNPs) Human Cells Processing 1. 5’-end of 45S precursor RNA is removed yielding the 41S 2. 41S precursor is cut into 2 parts: 1. 20S precursor of 18S 2. 32S precursor of 5.8S and 28S rRNA 3. 3’-end of 20S precursor is removed, yielding mature 18S rRNA 4. 32S precursor is cut to liberate 5.8S and 28S rRNA 5. 5.8S and 28S rRNA associate by base-pairing Bacterial rRNA Processing • Bacterial rRNA precursors contain tRNAs and all 3 rRNA • rRNA are released from their precursors by RNase III and RNase E – RNase III is the enzyme that performs at least the initial cleavages that separate the individual large rRNAs – RNase E is another ribonuclease that is responsible for removing the 5S rRNA from the precursor Processing Bacterial rRNA Spacers surrounding individual rRNAs genes are complementary and can form an extended hairpin; the double stranded region will serve as a target for RNAase III Transfer RNA Processing • Transfer RNAs are made in all cells as long precursors – These must be processed by removing RNA at both ends • Nuclei of eukaryotes contain precursors of a single tRNA • In bacteria, precursor may contain one or more tRNA, sometimes in combination with rRNAs Polycistronic Precursors • In processing bacterial RNA that contain more than one tRNA – First step is to cut precursor up into fragments with just one tRNA each • Cutting between tRNAs in precursors having two or more tRNA • Cutting between tRNAs and rRNAs in precursors – Enzyme that performs both chores is the RNase III 5’-Ends Maturation – Extra nucleotides are removed from the 5’-ends of pre-tRNA in one step by an endonucleolytic cleavage catalyzed by RNase P – RNase P from bacteria and eukaryotic nuclei have a catalytic RNA subunit called M1 RNA – Spinach chloroplast RNase P appears to lack an RNA subunit 5’-Ends Maturation – RNase P makes a cut at the site that becomes mature 5’-end of a tRNA – This enzyme is all that is needed to form mature 5’-ends 3’-Ends Maturation • Six RNases participate in 3’ ends processing: RNase D, RNase BN, RNase T, RNase PH, RNase II and polynucleotide phosphorylase (PNPase) • RNase II and polynucleotide phosphorylase cooperate in removing most of 3’-trailer from the end of a tRNA precursor – Stop at the +2 stage, with 2 extra nucleotides remaining at the 3’ end of tRNA • RNases PH and T remove the last two nucleotides from RNA – RNase T is the major participant in removing very last nucleotide 3’-Ends Maturation Trans-Splicing – Splicing that occurs in all eukaryotic species is called cis-splicing because it involves two or more exons that exist together in the same gene – Alternatively, trans-splicing has exons that are not part of the same gene at all, may not even be on the same chromosome Trans-Splicing – First discovered in trypanosomes – Trypanosome mRNA has 35 extra nucleotides that are missing from the gene – All Trypanosome mRNA have the same 35bp sequence, spliced leader (SL) on the 5’ end – The SL is encoded by a separate gene that is repeated over 200 times in a genome – The SL gene is composed of a short SL exon followed by 5’ part of intron Joining the SL to the Coding Region of an mRNA Priming Trans-splicing Trans-Splicing Scheme – Branchpoint adenosine within the half-intron attached to the coding exon attacks the junction between the leader exon and its half-intron – Creates a Y-shaped intronexon intermediate analogous to the lariat intermediate 9 Formation of Y-shaped intermediate confirms the second, trans-splicing hypothesis Posttranscriptional RNA Modification – Editing RNA editing – is a posttranscriptional changes of RNA nucleotide sequence involving: – Insertions – Deletions – Substitutions (conversions) Versus Capping, splicing, 3’ end formation, trimming of ends, modification of bases (e.g. methylation) etc. are RNA processing. Two Mechanisms of RNA Editing Substitution Editing: – chemical alteration of individual nucleotides (the equivalent of point mutations) – catalyzed by enzymes that recognize a specific target sequence of nucleotides: – cytidine deaminase that converts a C in the RNA to uracil (U) – adenosine deaminase that converts an A to inosine (I), which the ribosome translates as a G (the CAG codon (for Gln) can be converted to a CGG codon (for Arg) Two Mechanisms of RNA Editing Insertion/Deletion Editing: – insertion or deletion of nucleotides in RNA – mediated by guide RNA molecules that serve as a template for the addition and/or removal of nucleotides in the target RNA RNA Editing – Cryptogenes are incomplete genes – Trypanosomatid mitochondria, kinetoplasts, encode incomplete mRNA that must be edited before being translated – Editing occurs in the 3’→5’ direction by successive action of one or more guide RNAs RNA Editing ¾ Trypanosomatid kinetoplasts contain two types of circular DNA linked together – Maxicircles (contain mitochondrial genes, encode gRNAs) – Minicircles (involved in control of mitochondrial gene expression) RNA Editing – The U’s added by editing are shown in grey; the T’s present in a gene but absent in the mRNA are shown in blue – 731-nt fragment of COIII mRNA contains 407 UMPs added by editing; 19 UPMs originally coded by gene were removed from mRNA. gRNAs The gRNAs have three main regions –Anchor region – a 5' sequence that is complementary to the premRNA just 3' of the editing site –Guiding region – 3’ sequence that provides information needed for editing of pre-mRNA; the G is allowed to pair with U as well as with C nucleotides –3' oligo[U] tail – non-encoded sequence of U nucleotides 9 gRNA would form a perfect duplex with edited mRNA except for the oligo[U] tail. Mechanism of Editing • Unedited transcripts can be found along with edited versions of the same mRNAs • Editing occurs in the poly(A) tails of mRNAs that are added posttranscriptionally – Editing is a posttranscriptional modification • Partially edited transcripts have been isolated; they were always edited at their 3’-ends but not at their 5’-ends – Editing goes in the 3’→5’ direction gRNA and Editing – Guide RNAs (gRNA) could direct the insertion and deletion of UMPs over a stretch of nucleotides in the mRNA – 5’-end of gRNA is complementary to the region of pre-mRNA that requires no editing; 3’-end directs the editing of premRNA. Editing starts at 3’-end of premRNA – When editing is done, the new gRNA may hybridize near the 5’-end of newly edited region and continue the editing – The editing progressively continues in the direction of the 5’-end of pre-mRNA RNA Editing – gRNAs provide A’s and G’s as templates for the incorporation of U’s missing from the mRNA – If gRNA is missing an A or G to pair with a U in the pre-mRNA, then the U is removed ¾ Formation of weak G-U pairs enables displacement of 3’ end of an old gRNA by 5’ end of a new gRNA that base pairs with the newly edited region Removing and Adding U’s • Mechanism of removing U’s involves – Cutting pre-mRNA just beyond U to be removed – Removal of U by exonuclease – Ligating the two pieces of pre-mRNA together • Mechanism of adding U’s uses same first and last step – Middle step involves addition of one or more U’s from UTP by terminal uridylyl transferase (TUTase) Examples of RNA Editting Type of RNA editing Posttransciptional insertion and deletion of nucleotides. Cotranscriptional insertion of nucleotides Modification – substitution of Base Mechanism Genes and Organism U Guide RNAs Mitochondrial genes of Trypanosomes C ? Mitochondrial genes of Physarum polycephalum A Polyadenylation Mitochondrial genes of vertebrates G Polymerase stuttering Phosphoprotein gene of Paramyxoviruses C Deamination (to U) Apolipoprotein gene of mammals. Mitochondrial and chloroplast genes of plants U (to C) Wilm’s tumor susceptibility gene, WT1 of mammals A (to I) Mammalian glutamate receptor genes, matrix gene of Measles virus. nucleotides Conversions Insertion/deletions Examples of RNA Editting 1. Transcipts of trypanosomes 2. Mitochondria of P. polycephalum 3. Paramyxoviruses (P gene) 4. Mitochondria of vertebrates 5. Apolipoprotein B gene of mammals 6. Mitochondria of higher plants 7. Chloroplasts of higher plants 8. Glutamate-gated ion channels of mammals ..AAUUUAUGUUGUCUUU ..AUCUCUAAGGGUUUAACCGG Frameshift ..AUUAAAAAGGGGCACAC… Frameshift ..CAGUAAAAAAAAAA… Stop C ..GAUAUAAUUUGAUCAGUAUA… C Stop C ..UUUUUCAUUGUGGUUUAC… PHE TYR C ..AAUAAUAUGGCGAAACAU… Start A ..UUUAUGCGGCAAGGA… ARG Reading: R. Weaver, Molecular Biology, 4th ed. Chapter 16: pages 481-494.