Chapter 21 RNA Splicing and Processing

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Chapter 21 RNA Splicing and Processing Powered By Docstoc
					        Chapter 21
RNA Splicing and Processing
                21.1 Introduction

• pre-mRNA –The nuclear transcript that is processed by
  modification and splicing to give an mRNA.
• RNA splicing – The process of excising introns from
  RNA and connecting the exons into a continuous mRNA.
FIGURE 01: Eukaryotic mRNA is modified, processed, and transported
                21.1 Introduction

• heterogeneous nuclear RNA (hnRNA) – RNA
  that comprises transcripts of nuclear genes
  made by RNA polymerase II; it has a wide size
  distribution and low stability.
• hnRNP – The ribonucleoprotein form of hnRNA
  (heterogeneous nuclear RNA), in which the
  hnRNA is complexed with proteins.
  – Pre-mRNAs are not exported until processing is
    complete; thus they are found only in the nucleus.
   21.2 The 5′ End of Eukaryotic mRNA Is
                  Capped
• A 5′ cap is formed by adding a G to the terminal base of
  the transcript via a 5′–5′ link.
• The capping process takes place during the
  transcription, which may be important for transcription
  reinitiation.




 FIGURE 02: Eukaryotic mRNA
    has a methylated 5’ cap
   21.2 The 5′ End of Eukaryotic mRNA Is
                  Capped
• The 5′ cap of most mRNA is monomethylated, but some
  small noncoding RNAs are trimethylated.
• The cap structure is recognized by protein factors to
  influence mRNA stability, splicing, export, and
  translation.
   21.3 Nuclear Splice Junctions Are Short
                Sequences
• Splice sites are the sequences immediately surrounding
  the exon–intron boundaries. They are named for their
  positions relative to the intron.
• The 5′ splice site at the 5′ (left) end of the intron includes
  the consensus sequence GU.
• The 3′ splice site at the 3′ (right) end of the intron
  includes the consensus sequence AG.
21.3 Nuclear Splice Junctions Are Short Sequences
• The GU-AG rule (originally called the GT-AG rule in
  terms of DNA sequence) describes the requirement for
  these constant dinucleotides at the first two and last two
  positions of introns in pre-mRNAs.




                                         FIGURE 03: The ends of
                                        nuclear introns are defined
                                            by the GU-AG rule
  21.3 Nuclear Splice Junctions Are Short
               Sequences
• There exist minor introns relative to the major introns
  that follow the GU-AG rule.
• Minor introns follow a general AU-AC rule with a different
  set of consensus sequences at the exon–intron
  boundaries.
   21.4 Splice Junctions Are Read in Pairs

• Splicing depends only on recognition of pairs of splice
  junctions.
• All 5′ splice sites are functionally equivalent, and all 3′
  splice sites are functionally equivalent.
• Additional conserved sequences at both 5′ and 3′ splice
  sites define functional splice sites among numerous
  other potential sites in the pre-mRNA.
FIGURE 04: Correct splicing removes three introns by pairwise recognition
                             of the junctions
21.5 Pre-mRNA Splicing Proceeds through
               a Lariat
• Splicing requires the 5′ and 3′ splice sites and a branch
  site just upstream of the 3′ splice site.
• The branch sequence is conserved in yeast but less well
  conserved in multicellular eukaryotes.
• A lariat is formed when the intron is cleaved at the 5′
  splice site, and the 5′ end is joined to a 2′ position at an A
  at the branch site in the intron.
21.5 Pre-mRNA Splicing Proceeds through
               a Lariat

                  • The intron is released as a
                    lariat when it is cleaved at
                    the 3′ splice site, and the left
                    and right exons are then
                    ligated together.



                     FIGURE 05: Splicing proceeds
                           through a lariat
   21.6 snRNAs Are Required for Splicing

• small cytoplasmic RNAs (scRNA; scyrps) – RNAs
  that are present in the cytoplasm (and sometimes are
  also found in the nucleus).
• small nuclear RNA (snRNA; snurps) – One of many
  small RNA species confined to the nucleus; several of
  them are involved in splicing or other RNA processing
  reactions.
• small nucleolar RNA (snoRNA) – A small nuclear RNA
  that is localized in the nucleolus.
   21.6 snRNAs Are Required for Splicing

• The five snRNPs involved in splicing are U1, U2, U5, U4,
  and U6.
• Together with some additional proteins, the snRNPs form
  the spliceosome.




                               FIGURE 07: The spliceosome
                                    is a large particle
   21.6 snRNAs Are Required for Splicing

• All the snRNPs except U6 contain a conserved
  sequence that binds the Sm proteins that are recognized
  by antibodies (anti-SM) generated in autoimmune
  disease.
• splicing factor – A protein component of the
  spliceosome that is not part of one of the snRNPs.
• transesterification – A reaction that breaks and makes
  chemical bonds in a coordinated transfer so that no
  energy is required.
    21.7 Commitment of Pre-mRNA to the
            Splicing Pathway
• U1 snRNP initiates splicing by binding to the 5′ splice
  site by means of an RNA–RNA pairing reaction.
• The commitment complex (or E complex) contains U1
  snRNP bound at the 5′ splice site and the protein U2AF
  bound to a pyrimidine tract between the branch site and
  the 3′ splice site.
FIGURE 10: Formation of the commitment complex
    21.7 Commitment of Pre-mRNA to the
            Splicing Pathway
• In multicellular eukaryotic cells, SR proteins play an
  essential role in initiating the formation of the
  commitment complex.
• Pairing splice sites can be accomplished by intron
  definition or exon definition.
FIGURE 11: Two route for initial recognition of 5’ and 3’ splice sites
 21.8 The Spliceosome Assembly Pathway

• The commitment complex progresses to pre-
  spliceosome (the A complex) in the presence of ATP.
• Recruitment of U5 and U4/U6 snRNPs converts the pre-
  spliceosome to the mature spliceosome (the B1
  complex).
• The B1 complex is next converted to the B2 complex in
  which U1 snRNP is released to allow U6 snRNA to
  interact with the 5′ splice site.
21.8 The Spliceosome Assembly Pathway
                                    • U4 dissociates from U6
                                      snRNP to allow U6
                                      snRNA to pair with U2
                                      snRNA to form the
                                      catalytic center for
                                      splicing.
                                    • Both transesterification
                                      reactions take place in
                                      the activated spliceosome
                                      (the C complex).
                                    • The splicing reaction is
                                      reversible at all steps.

  FIGURE 12: Splicing reaction proceeds through discrete stages
   21.9 An Alternative Spliceosome Uses
   Different snRNPs to Process the Minor
               Class of Introns
• An alternative splicing pathway uses another set of
  snRNPs with only U5 snRNP in common with the major
  spliceosome.
• The target introns are defined by longer consensus
  sequences at the splice junctions, rather than strictly
  according to the GU-AG or AU-AC rules.
• Major and minor spliceosomes share critical protein
  factors, including SR proteins.
21.10 Pre-mRNA Splicing Likely Shares the
   Mechanism with Group II Autocatalytic
                 Introns
• Group II introns excise themselves from RNA by an
  autocatalytic splicing event (autosplicing or self-
  splicing).
• The splice junctions and mechanism of splicing of group
  II introns are similar to splicing of nuclear introns.
• A group II intron folds into a secondary structure that
  generates a catalytic site resembling the structure of U6-
  U2-nuclear intron.
FIGURE 15: Splicing uses transesterification
     21.11 Splicing Is Temporally and
 Functionally Coupled with Multiple Steps in
              Gene Expression
• Splicing can occur during or after transcription.
• The transcription and splicing machineries are physically
  and functionally integrated.
• Splicing is connected to mRNA export and stability
  control.
• exon junction complex (EJC) – A protein complex that
  assembles at exon–exon junctions during splicing and
  assists in RNA transport, localization, and degradation.
FIGURE 18: Splicing is required for mRNA export
    21.11 Splicing Is Temporally and
Functionally Coupled with Multiple Steps in
             Gene Expression
                    • Splicing in the nucleus can
                      influence mRNA translation in
                      the cytoplasm.
                    • nonsense-mediated mRNA
                      decay (NMD) – A pathway
                      that degrades an mRNA that
                      has a nonsense mutation
                      prior to the last exon.


                       FIGURE 20: The EJC complex
                         couples splicing with NMD
21.12 Alternative Splicing Is a Rule, Rather
    Than an Exception, in Multicellular
                Eukaryotes
• Specific exons or exonic sequences may be excluded or
  included in the mRNA products by using alternative
  splicing sites.
• Alternative splicing contributes to structural and
  functional diversity of gene products.
FIGURE 21: Different modes of alternative splicing.
21.12 Alternative Splicing Is a Rule, Rather
    Than an Exception, in Multicellular
                Eukaryotes
• Sex determination in Drosophila involves a series of
  alternative splicing events in genes coding for
  successive products of a pathway.
FIGURE 23: Sex determination in D. melanogaster
21.13 Splicing Can Be Regulated by Exonic
    and Intronic Splicing Enhancers and
                  Silencers
• Alternative splicing is often associated with weak splice
  sites.
• Sequences surrounding alternative exons are often more
  evolutionarily conserved than sequences flanking
  constitutive exons.
• Specific exonic and intronic sequences can enhance or
  suppress splice site selection.
FIGURE 24: Exonic and intronic sequences can modulate the splice site
                              selection
 21.13 Splicing Can Be Regulated by Exonic
and Intronic Splicing Enhancers and Silencers
• The effect of splicing enhancers and silencers is
  mediated by sequence-specific RNA binding proteins,
  many of which may be developmentally regulated and/or
  expressed in a tissue-specific manner.
• The rate of transcription can directly affect the outcome
  of alternative splicing.



  FIGURE 25: The Nova
 and Fox families of RNA
     binding proteins
  21.14 trans-Splicing Reactions Use Small
                    RNAs
                                      • Splicing reactions usually
                                        occur only in cis between
                                        splice junctions on the same
                                        molecule of RNA.
                                      • trans-splicing occurs in
                                        trypanosomes and worms
                                        where a short sequence (SL
                                        RNA) is spliced to the 5′ ends
                                        of many precursor mRNAs.
                                      • SL RNAs have a structure
                                        resembling the Sm-binding
                                        site of U snRNAs.
FIGURE 26: Trans-splicing can occur
 when special constructs are made
        21.15 The 3′ Ends of mRNAs Are
          Generated by Cleavage and
                Polyadenylation
                                • The sequence AAUAAA is a
                                  signal for cleavage to generate
                                  a 3′ end of mRNA that is
                                  polyadenylated.
                                • The reaction requires a protein
                                  complex that contains a
                                  specificity factor, an
                                  endonuclease, and poly(A)
                                  polymerase.
                                • The specificity factor and
  FIGURE 29: The 3’ end of
                                  endonuclease cleave RNA
mRNA is generated by cleavage     downstream of AAUAAA.
21.15 The 3′ Ends of mRNAs Are
  Generated by Cleavage and
        Polyadenylation
                          • The specificity factor and
                            poly(A) polymerase add
                            ~200 A residues
                            processively to the 3′ end.
                          • The poly(A) tail controls
                            mRNA stability and
                            influences translation.
                          • Cytoplasmic polyadenylation
                            plays a role in Xenopus
                            embryonic development.

FIGURE 30: There is a single 3’ end-processing complex
21.16 The 3′ mRNA End Processing Is
 Critical for Transcriptional Termination
                     • There are various ways to
                       end transcription by
                       different RNA
                       polymerases.
                     • The mRNA 3′ end
                       formation signals
                       termination of Pol II
                       transcription.

                      FIGURE 31: Transcription by Pol I
                               and Pol III
   21.17 The 3′ End Formation of Histone
        mRNA Requires U7 snRNA
• The expression of histone mRNAs is replication
  dependent and is regulated during the cell cycle.
• Histone mRNAs are not polyadenylated; their 3′ ends are
  generated by a cleavage reaction that depends on the
  structure of the mRNA.
   21.17 The 3′ End Formation of Histone
        mRNA Requires U7 snRNA
• The cleavage reaction requires the SLBP to bind to a
  stem-loop structure and the U7 snRNA to pair with an
  adjacent single-stranded region.
• The cleavage reaction is catalyzed by a factor shared
  with the polyadenylation complex.
FIGURE 33: Generation of the 3’ end of histone H3 mRNA
 21.18 tRNA Splicing Involves Cutting and
     Rejoining in Separate Reactions
• RNA polymerase III terminates transcription in a poly(U)4
  sequence embedded in a GC-rich sequence.
• tRNA splicing occurs by successive cleavage and
  ligation reactions.



                                    FIGURE 34: tRNA splicing
                                  recognized a specific structure
  21.18 tRNA Splicing Involves Cutting and
      Rejoining in Separate Reactions
• An endonuclease cleaves the tRNA precursors at both
  ends of the intron.
• Release of the intron generates two half-tRNAs with
  unusual ends that contain 5′ hydroxyl and 2′–3′ cyclic
  phosphate.




 FIGURE 36: The endonuclease
   complex has four proteins
 21.18 tRNA Splicing Involves Cutting and
     Rejoining in Separate Reactions
• The 5′–OH end is phosphorylated by a polynucleotide
  kinase, the cyclic phosphate group is opened by
  phosphodiesterase to generate a 2′–phosphate terminus
  and 3′–OH group, exon ends are joined by an RNA
  ligase, and the 2′–phosphate is removed by a
  phosphatase.
FIGURE 38: tRNA splicing has separate cleavage and ligation stages
  21.19 The Unfolded Protein Response Is
         Related to tRNA Splicing
• Ire1 is an inner nuclear membrane protein with its N-
  terminal domain in the ER lumen and its C-terminal
  domain in the nucleus; the C-terminal domain exhibits
  both kinase and endonuclease activities.
• Binding of an unfolded protein to the N-terminal domain
  activates the C-terminal endonuclease by
  autophosphorylation.
FIGURE 39: Unfolded proteins activate a TF
  21.19 The Unfolded Protein Response Is
         Related to tRNA Splicing
• The activated endonuclease cleaves HAC1 (Xbp1 in
  vertebrates) mRNA to release an intron and generate
  exons that are ligated by a tRNA ligase.
• Only spliced HAC1 mRNA can be translated to a
  transcription factor that activates genes coding for
  chaperones that help to fold unfolded proteins.
• Activated Ire1 induces apoptosis when the cell is over
  stressed by unfolded proteins.
    21.20 Production of rRNA Requires
 Cleavage Events and Involves Small RNAs
• RNA polymerase I terminates transcription at an 18-base
  terminator sequence.
• The large and small rRNAs are released by cleavage
  from a common precursor rRNA; the 5S rRNA is
  separately transcribed.




         FIGURE 40: Generation of mature eukaryotic rRNAs
   21.20 Production of rRNA Requires
Cleavage Events and Involves Small RNAs
                                   • The C/D group of snoRNAs is
                                     required for modifying the 2′
                                     position of ribose with a methyl
                                     group.
                                   • The H/ACA group of snoRNAs
                                     is required for converting
                                     uridine to pseudouridine.
                                   • In each case the snoRNA base
                                     pairs with a sequence of rRNA
 FIGURE 42: A snoRNA base            that contains the target base to
pairs with a region of rRNA that     generate a typical structure that
      is to be methylated
                                     is the substrate for modification.

				
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