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RNA processing

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									Transcription and Post-transcription
            Modification



             李 希
      分子医学教育部重点实验室
        lixi@shmu.edu.cn
Post-transcriptional
Processing of RNA

Making ends of RNA
   RNA splicing
             Primary Transcript


• Primary Transcript-the initial molecule of RNA
  produced--- hnRNA (heterogenous nuclear
  RNA )
• In prokaryotes, DNA →RNA →protein in
  cytoplasm concurrently
• In eukaryotes nuclear RNA >> Cp RNA
Processing of eukaryotic pre-mRNA



                             For primary transcripts
                             containing multiple
                             exons and introns,
                             splicing occurs before
                             transcription of the gene
                             is complete--co-
                             transcriptional splicing.




                        Human dystrophin gene has 79
                        exons, spans over 2,300-Kb and
                        requires over 16 hours to be
                        transcribed!
        Types of RNA processing
A) Cutting and trimming to generate ends:
   rRNA, tRNA and mRNA
B) Covalent modification:
   Add a cap and a polyA tail to mRNA
   Add a methyl group to 2’-OH of ribose in mRNA and
     rRNA
   Extensive changes of bases in tRNA
C) Splicing
   pre-rRNA, pre-mRNA, pre-tRNA by different
     mechanisms.
The RNA Pol II CTD is required for the coupling of transcription with
         mRNA capping, polyadenylation and splicing

                                                  1.   The coupling allows
                                                       the processing
                                                       factors to present at
                                                       high local
                                                       concentrations
                                                       when splice sites
                                                       and poly(A) signals
                                                       are transcribed by
                                                       Pol II, enhancing the
                                                       rate and specificity
                                                       of RNA processing.

                                                  2.   The association of
                                                       splicing factors with
                                                       phosphorylated CTD
                                                       also stimulates Pol II
                                                       elongation. Thus, a
                                                       pre-mRNA is not
                                                       synthesized unless
                                                       the machinery for
                                                       processing it is
                                                       properly positioned.
Time course of RNA processing
   5’ and 3’ ends of eukaryotic mRNA




           5’-UTR     3’-UTR

Add a GMP                      Cut the pre-mRNA
Methylate it and               and add A’s
1st few nucleotides
            Capping of pre-mRNAs

• Cap=modified guanine nucleotide
• Capping= first mRNA processing event - occurs during
  transcription
• CTD recruits capping enzyme as soon as it is
  phosphorylated
• Pre-mRNA modified with 7-methyl-guanosine
  triphosphate (cap) when RNA is only 25-30 bp long
• Cap structure is recognized by CBC(cap-binding
  complex)
      •   stablize the transcript
      •   prevent degradation by exonucleases
      •   stimulate splicing and processing
Capping of the 5’ end of nascent RNA transcripts with m7G
                                              • The  cap is added
                                              after the nascent RNA
                                              molecules produced
                                              by RNA polymerase II
                                              reach a length of 25-
                                              30 nucleotides.
                      Existing in
                                              Guanylyltransferase is
                      a single                recruited and activated
                      complex
                                              through binding to the
                                              Ser5-phosphorylated
                                              Pol II CTD.

                                              • The methyl groups
                                              are derived from S-
                                              adenosylmethionine.
           Sometimes
           methylated
                                              • Capping helps
                                              stabilize mRNA and
                                              enhances translation,
                                              splicing and export
         Sometimes
         methylated                           into the cytoplasm.
Consensus sequence for 3’ process




AAUAAA: CstF (cleavage stimulation factor F)
GU-rich sequence: CPSF (cleavage and polyadenylation
specificity factor)
Polyadenylation of mRNA at the 3’ end
CPSF: cleavage and polyadenylation specificity
factor.
CStF: cleavage stimulatory factor.
CFI & CFII: cleavage factor I & II.
PAP: poly(A) polymerase.
PABPII: poly(A)-binding protein II.
RNA is cleaved 10~35-nt 3’ to A2UA3.
The binding of PAP prior to cleavage ensures
that the free 3’ end generated is rapidly
polyadenylated.
PAP adds the first 12A residues to 3’-OH
slowly.
Binding of PABPII to the initial short
poly(A) tail accelerates polyadenylation by
PAP.
Poly(A) tail stabilizes mRNA and enhances
translation and export into the cytoplasm.
The polyadenylation complex is associated with
the CTD of Pol II following initiation.
        Functions of 5’ cap and 3’ polyA

• Need 5’ cap for efficient translation:
   – Eukaryotic translation initiation factor 4 (eIF4)
     recognizes and binds to the cap as part of initiation.
• Both cap and polyA contribute to stability of mRNA:
   – Most mRNAs without a cap or polyA are degraded
     rapidly.
   – Shortening of the polyA tail and decapping are part of
     one pathway for RNA degradation in yeast.
             mRNA Half-life

• t½ seconds if seldom needed
• t½ several cell generations (i.e. ~48-72 h) for
   houskeeping gene
 • ≈avg 3 h in eukaryotes
 • ≈avg 1.5 min in bacteria
Poly(A)+ RNA can be separated from other RNAs
by fractionation on Sepharose-oligo(dT).
Split gene and mRNA splicing
    The discovery of split genes (1977)
       1993 Noble Prize in Medicine
To Dr. Richard Robert and Dr. Phillip Sharp
             Background: Adenovirus has a DNA genome and
             makes many mRNAs. Can we determine which
             part of the genome encodes for each mRNA by
             making a DNA:RNA hybrid?

             Experiment: Isolate Adenovirus genomic DNA,
             isolate one adenovirus mRNA, hybridize and
             then look by EM at where the RNA hybridizes
             (binds) to the genomic DNA.

             Surprise: The RNA is generated from 4
             different regions of the DNA! How can we
             explain this? Splicing!!
                         mRNA




           DNA



The matured mRNAs are much shorter than
the DNA templates.
            Exon and Intron

• Exon is any segment of an interrupted
  gene that is represented in the mature RNA
  product.
• Intron is a segment of DNA that is
  transcribed, but removed from within the
  transcript by splicing together the
  sequences (exons) on either side of it.
Exons are
similar in size




Introns are highly
variable in size
                               GT-AG rule




• GT-AG rule describes the presence of these constant dinucleotides at the first
 two and last two positions of introns of nuclear genes.
• Splice sites are the sequences immediately surrounding the exon-intron boundaries
• Splicing junctions are recognized only in the correct pairwise combinations
 The sequence of steps in the production of mature eukaryotic mRNA as shown
                        for the chicken ovalbumin gene.




The consensus sequence at the exon–intron junctions of vertebrate pre-mRNAs.
           4 major types of introns
     4 classes of introns can be distinguished on the basis
of their mechanism of splicing and/or characterisitic
sequences:
– Group I introns in fungal mitochondria, plastids,
  and in pre-rRNA in Tetrahymena (self-splicing)
– Group II introns in fungal mitochondria and
  plastids (self-splicing)
– Introns in pre-mRNA (spliceosome mediated)
– Introns in pre-tRNA
Group I and II introns
The sequence of transesterification reactions that
splice together the exons of eukaryotic pre-mRNAs.
      Splicing of Group I and II introns
• Introns in fungal mitochondria, plastids, Tetrahymena pre-
  rRNA
• Group I
   – Self-splicing
   – Initiate splicing with a G nucleotide
   – Uses a phosphoester transfer mechanism
   – Does not require ATP hydrolysis.
• Group II
   – self-splicing
   – Initiate splicing with an internal A
   – Uses a phosphoester transfer mechanism
   – Does not require ATP hydrolysis
   Self-splicing in pre-rRNA in Tetrahymena :
                T. Cech et al. 1981

                                                  +
 Exon 1    Intron 1 Exon 2        Exon 1 Exon 2       Intron 1

•Products of splicing were resolved by gel electrophoresis:
            pre-rRNA +        +    +     +
      Nuclear extract -       +     -     +        Additional proteins
                  GTP -       +    +     -         are NOT needed for
                                                  splicing of this pre-
         pre-rRNA
                                                  rRNA!
     Spliced exon
                                                  Do need a G
      Intron circle                               nucleotide (GMP,
      Intron linear                               GDP, GTP or
                                                  Guanosine).
The sequence of reactions in the self-splicing of
Tetrahymena group I intron.
Where is the catalytic activity in RNase P?

 RNase P is composed of a 375 nucleotide RNA and
 a 20 kDa protein. The protein component will NOT
 catalyze cleavage on its own.

 The RNA WILL catalyze cleavage by itself !!!!
 The protein component aids in the reaction but is not
 required for catalysis.
 Thus RNA can be an enzyme.

 Enzymes composed of RNA are called ribozymes.
          Hammerhead ribozymes
• A 58 nt structure is used in self-cleavage
• The sequence CUGA adjacent to stem-
  loops is sufficient for cleavage
              3'                  5'

                       CG
                       U A             Bond that is cle av e d.
                       C G
                       AUC
               AA                 ACCAC
              A
         GGCC                   C UGGUG
         CCGG A             U
                  GU   AG
                                CUGA is r e quire d for catalysis
Mechanism of hammerhead ribozyme

• The folded RNA forms an active site for
  binding a metal hydroxide
• Attracts a proton from the 2’ OH of the
  nucleotide at the cleavage site.
• This is now a nucleophile for attack on the 3’
  phosphate and cleavage of the
  phosphodiester bond.


                1989 Nobel Prize in chemistry, Sidney Altman,
                and Thomas Cech
       Distribution of Group I introns

• Prokaryotes – eubacteria (tRNA & rRNA), phage
• Eukaryotes
   – lower (algae, protists, & fungi)
       • nuclear rRNA genes, organellar genes, Chlorella
         viruses
   – higher plants: organellar genes
   – lower animals (Anthozoans): mitochondrial

• >1800 known, classified into ~12 subgroups, based on
  secondary structure
            Splicing of pre-mRNA

• The introns begin and end with almost invariant
  sequences: 5’ GU…AG 3’
• Use ATP hydrolysis to assemble a large
  spliceosome (45S particle, 5 snRNAs and 65
  proteins, same size and complexity as ribosome)
• Mechanism is similar to that of the Group II fungal
  introns:
   – Initiate splicing with an internal A
   – Uses a phosphoester transfer mechanism for
     splicing
  Initiation of phosphoester transfers in pre-mRNA
• Uses 2’ OH of an A internal to the
  intron
• Forms a branch point by attacking
  the 5’ phosphate on the first
  nucleotide of the intron
• Forms a lariat structure in the
  intron
• Exons are joined and intron is
  excised as a lariat
• A debranching enzyme cleaves the
  lariat at the branch to generate a
  linear intron
• Linear intron is degraded
     Involvement of snRNAs and snRNPs


• snRNAs = small nuclear RNAs
• snRNPs = small nuclear ribonucleoproteins
  particles (snRNA complex with protein)
• Addition of these antibodies to an in vitro pre-
  mRNA splicing reaction blocked splicing.
• Thus the snRNPs were implicated in splicing
   Role of snRNPs in RNA splicing


• Recognizing the 5’ splice site and the branch site.
• Bringing those sites together.
• Catalyzing (or helping to catalyze) the RNA cleavage.




   RNA-RNA, RNA-protein and protein-protein
   interactions are all important during splicing
                 snRNPs

U1, U2, U4/U6, and U5 snRNPs
– Have snRNA in each: U1, U2, U4/U6, U5
– Conserved from yeast to human
– Assemble into spliceosome
– Catalyze splicing
    Splicing of pre-mRNA occurs in a
 “spliceosome” an RNA-protein complex



           spliceosome
     (~100 proteins + 5 small RNAs)




pre-mRNA                            spliced mRNA

The spliceosome is a large protein-RNA complex
in which splicing of pre-mRNAs occurs.
            Assembly of spliceosome
• snRNPs are assembled progressively into the
  spliceosome.
  – U1 snRNP binds (and base pairs) to the 5’ splice site
  – BBP (branch-point binding protein) binds to the branch site
  – U2 snRNP binds (and base pairs) to the branch point, BBP
    dissociates
  – U4U5U6 snRNP binds, and U1 snRNP dissociates
  – U4 snRNP dissociates
• Assembly requires ATP hydrolysis
• Assembly is aided by various auxiliary factors and
  splicing factors.
Some RNA-RNA hybrids formed
during the splicing reaction


               Steps of the spliceosome-
               mediated splicing reaction
               Assembly of spliceosome




A schematic diagram of six rearrangements that the spliceosome undergoes in
mediating the first transesterification reaction in pre-mRNA splicing.
The spliceosome cycle
   The Significance of Gene Splicing

• The introns are rare in prokaryotic structural
  genes
• The introns are uncommon in lower eukaryote
  (yeast), 239 introns in ~6000 genes, only one
  intron / polypeptide
• The introns are abundant in higher eukaryotes
  (lacking introns are histons and interferons)
• Unexpressed sequences constitute ~80% of a
  typical vertebrate structural gene
Errors produced by mistakes
   in splice-site selection
     Mechanisms prevent splicing error
• Co-transcriptional loading process
• SR proteins recruit spliceosome components to the 5’ and
  3’ splice sites




• SR protein = Serine Arginine rich protein
• ESE = exonic splicing enhancers
• SR protein regulates alternative splicing
                Alternative splicing
• Alternative splicing occurs in all metazoa and is
  especially prevalent in vertebrate




        Five ways to splice an RNA
Regulated alternative splicing

                       Different signals in
                       the pre-mRNA and
                       different proteins
                       cause spliceosomes
                       to form in particular
                       positions to give
                       alternative splicing
Alternative splicing can generate mRNAs encoding
  proteins with different, even opposite functions
                                         Fas ligand
                                         Fas
                    5 6 7                        (membrane-
                                                 associated)




 Fas pre-mRNA                            (+)


  5          6              7    APOPTOSIS (programmed
                                             cell death)

                                         (-)




                                         Fas ligand
                     5 7                 Soluble Fas
                                               (membrane)
Drosophila Dscam gene contains thousands
        of possible splice variants




Alternative possibilities for 4 exons leave a total number of possible
mRNA variations at 38,016. The protein variants are important for
wiring of the nervous system and for immune response.
          Cis- and Trans-Splicing




Cis-: Splicing in single RNA
Trans-: Splicing in two different RNAs
         Y-shaped excised introns (cis-: lariat)
        Occur in C. elegance and higher eukaryotes but it does in only a
        few mRNAs and at a very low level
        Same splicing mechanism is
        employed in trans-splicing
pre-mRNA splicing                 trans-mRNA splicing

                        spliced leader




   Spliced leader contains the cap structure!
                 RNA editing

• RNA editing is the process of changing the
  sequence of RNA after transcription.
• In some RNAs, as much as 55% of the nucleotide
  sequence is not encoded in the (primary) gene,
  but is added after transcription.
• Examples: mitochondrial genes in Trypanosomes
  (锥虫)
• Can add, delete or change nucleotides by editing
 Two mechanisms mediate editing



• Guide RNA-directed uridine insertion
  or deletion
• Site-specific deamination
Insertion and deletion of nucleotides by editing
• Uses a guide RNA
  (in 20S RNP =
  editosome) that is
  encoded elsewhere
  in the genome
• Part of the guide
  RNA is
  complementary to
  the mRNA in vicinity
  of editing

                         Trypanosomal RNA editing pathways.
                         (a) Insertion. (b) Deletion.
Mammalian example of editing




 The C is converted to U in intestine by a specific
   deaminating enzyme, not by a guide RNA.
        Cutting and Trimming RNA

• Can use endonucleases to cut at specific sites
  within a longer precursor RNA
• Can use exonucleases to trim back from the
  new ends to make the mature product
• This general process is seen in prokaryotes and
  eukaryotes for all types of RNA
The posttranscriptional processing of
           E. coli rRNA.
   RNase III cuts in stems of stem-loops

             16S rRNA            23S rRNA



RNase III




No apparent primary sequence specificity - perhaps RNase III
recognizes a particular stem structure.
     Eukaryotic rRNA Processing

• The primary rRNA transcript (~7500nt, 45S RNA)
  contains 18S, 5.8S and 28S

• Methylation
  occur mostly in rRNA sequence
  80%: O2-methylribose, 20%: bases (A or G)

• peudouridine
  95 U in rRNA in human are converted to Y’s
  may contribute rRNA tertiary stability
       Transfer RNA Processing

• Cloverleaf structure
• CCA: amino acid
  binding site
• Anticodon
• ~60 tRNA genes in E.
  coli




                         A schematic diagram of the tRNA
                          cloverleaf secondary structure.
         Endo- and exonucleases to generate
                    ends of tRNA




•   Endonuclease RNase P cleaves to generate the 5’ end.
•   Endonuclease RNase F cleaves 3 nucleotides past the mature 3’ end.
•   Exonuclease RNase D trims 3’ to 5’, leaving the mature 3’ end.
                Splicing of pre-tRNA
• Introns in pre-tRNA are very short (about 10-20
  nucleotides)
• Have no consensus sequences
• Are removed by a series of enzymatic steps:
   – Cleavage by an endonuclease
   – Phosphodiesterase to open a cyclic intermediate and
      provide a 3’OH
   – Activation of one end by a kinase (with ATP hydrolysis)
   – Ligation of the ends (with ATP hydrolysis)
   – Phosphatase to remove the extra phosphate on the
      2’OH (remaining after phosphodiesterase )
              Steps in splicing of pre-tRNA

                                 +
                                     OH 5’
                             P
              1. Endo-                       2. Phospho-
                         2’,3’ cyclic
              nuclease                       diesterase
                         phosphate
                                             3. Kinase (ATP)   Spliced
                                             4. Ligase (ATP)   tRNA
Intron of
10-20
                             +               5. Phosphatase
nucleotides



                    Excised intron
            CCA at 3’ end of tRNAs

• All tRNAs end in the sequence CCA.
• Amino acids are added to the CCA end during
  “charging” of tRNAs for translation.
• For most eukaryotic tRNAs, the CCA is added
  after transcription, in a reaction catalyzed by tRNA
  nucleotidyl transferase.
All of the four bases in tRNA can be modified
 Pathologies resulting from aberrant splicing
   can be grouped in two major categories
• Mutations affecting proteins that are involved in splicing
  Examples: Spinal Muscular Atrophy
                Retinitis Pigmentosa
                Myotonic Dystrophy

• Mutations affecting a specific messenger RNA and disturbing its
  normal splicing pattern
  Examples: β-Thalassemia
                Duchenne Muscular Dystrophy
                Cystic Fibrosis
                Frasier Syndrome
                Frontotemporal Dementia and Parkinsonism
                  Intron Advantage?

• One benefit of genes with introns is a phenomenon called
  alternative splicing

• A pre-mRNA with multiple introns can be spliced in
  different ways
   – This will generate mature mRNAs with different
      combinations of exons

• This variation in splicing can occur in different cell types or
  during different stages of development
             Intron Advantage?

• The biological advantage of alternative splicing
  is that two (or more) polypeptides can be
  derived from a single gene

• This allows an organism to carry fewer genes
  in its genome
Do you
believe?
 RNA Interference and Interference RNA


RNA interference or RNAi is a
remarkable process whereby small
noncoding RNA silence specific genes.
  - RNAi was first observed in plant in immune
response to viral pathgens.
 - MicroRNA regulate gene expression in
organisms from nematode to man.

                 Nobel Prize in Physiology or Medicine
                 2006, Andrew . Fire and Craig . Mello
    RNA Interference: A Mechanism for
       Silencing Gene Expression

1. Small dsRNA fragments can silence the expression of a
   matching gene. This is RNA interference (RNAi),
   recently discovered in C. elegans.
   a. Injecting dsRNA into adult worms results in specific loss of the
      corresponding mRNA in the worm and its progeny.
   b. RNAi also occurs in many other organisms, where it protects
      against viral infection and regulates developmental processes.
2. RNAi is highly specific and sensitive, with only a few
   molecules of dsRNA needed, making it an excellent
   research tool.
            Comparison of siRNA and miRNA


                siRNA                   miRNA

Precursor       Endogenous or           Endogenous transcript
                   exogenous dsRNA
Structure       dsRNA                   ssRNA

Function        mRNA cleavage           Translation inhibition and
                                           mRNA cleavage

Target mRNA     perfect complimentarity Imperfect complimentarity
                Inhibit transpon and    development
Biological
                   virus infection
Foreign DNA and Transgene

Foreign DNA and Transgene


 Aberrant sense RNA

              RdRP
                       dsRNA
              Dicer
                                 Nature 2004,
        siRNAs
                                 Vol 431,
                                 Sept.16:343

 Heterochromatin formation
 and Transcriptional silencing
                     Self splicing miRNA
 Mitrons :Short intronic
 hairpins
            RNA Ploymerase II or
            III
 pri-miRNA
                      No need of Drosha
                      Splicing machinery
                      Lariat debranching enzyme
 pre-miRNAs
                                                  Cell 130, July 13
             Dicer                                2007: 89-100

Micro RNAs (MiRNAs)
                                      RISC
~22NTs
lncRNA functions
Something I may not care , but you have to.

								
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