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RNA Processing

Soo-Chen Cheng

Institute of Molecular Biology


Academia Sinica

   
RNA Processing

Alteration of RNA molecules from their primary transcripts


to mature forms.

Trimming and addition - rRNA, tRNA, mRNA, snRNA

Base modification - rRNA, tRNA, snRNA

Splicing - rRNA, tRNA, mRNA, snRNA

Editing - rRNA, tRNA, mRNA

Degradation
   
Overview of mRNA Processing in Eukaryotes

   
Ribonucleoprotein Complexes

RNA molecules do not usually exist as free RNA


molecules. They are mostly associated with proteins
to form ribonucleoprotein particles (RNPs).

In nucleus
pre-mRNAs form hnRNPs for heterogeneous
nuclear ribonucleoprotein particles.

In cytoplasm
mRNAs form mRNPs.

Conserved RNA-binding motifs


RRM (RNA recognition motif), KH motif, RGG box
   
RNA Processing
• RNA Splicing

• 3’-End Formation of mRNA


• rRNA Processing and Modification

• RNA Editing

   
RNA Splicing

• Splicing of tRNA, nuclear rRNA,


nuclear mRNA and organelle RNA
• Alternative splicing regulation

   
RNA Splicing

Different classes of RNA splice their precursors through


different mechanisms due to difference in their structure.

tRNA -- Some tRNAs have introns


Enzymatic cleavage-ligation reaction

rRNA -- Tetrahymena 26S rRNA has an intron


RNA catalyzed transesterification - self-splicing

mRNA -- Most eukaryotic mRNAs have introns


Require complicated RNPs machinery to fold RNA

Organelle RNA -- Fungal mitochondrial mRNAs, rRNA or some


  cloroplast mRNAs
  and tRNAs have introns
Splicing of pre-tRNA

The structure of tRNA and Pre-tRNA

tRNA Pre-tRNA

   
Structure of tRNA and pre-tRNA

tRNA Pre-tRNA

   
Splicing of pre-tRNA
tRNA Endonuclease tRNA Ligase

Sen2 - 5’ splice site cleavage


Sen34 - 3’ splice site cleavage

   
Consensus Sequences around
5’ and 3’ Splice Sites of Pre-mRNA

The branch point is always an A.

Major

AT-AC

   
Chemistry of the Pre-mRNA Splicing Reaction

   
Factors Involved in Pre-mRNA Splicing

RNA Components

Small nuclear RNAs - U1, U2, U4, U5, U6


U11, U12, U4atac, U5, U6atac
Protein Components

   
Spliceosomal snRNAs

Lsm binding
U6 U5
U1 U2

U4
Sm binding Sm binding
Sm binding Sm binding

Lsm

   
Starket et al. Nature 409, 539-541 (2001) Collins et al. JMB 309, 915-923 (2001)
Base Pairing between U1, U2 and Pre-mRNA
Early in the Splicing Reaction

   
Factors Involved in Pre-mRNA Splicing

RNA Components

Small nuclear RNAs - U1, U2, U4, U5, U6


U11, U12, U4atac, U5, U6atac
Protein Components

Protein components of snRNPs

DExD/H-box RNA helicases (DEAD)

   
Factors Involved in Pre-mRNA Splicing

RNA Components

Small nuclear RNAs - U1, U2, U4, U5, U6


U11, U12, U4atac, U5, U6atac
Protein Components

Protein components of snRNPs

DExD/H-box RNA helicases (DEAD)

SR proteins

Others
   
SR Proteins in mRNA Splicing

Structural features: Binding sites:


Arginine/Serine rich domain Polypyrimidine tract
RRM (RNA recognition motif) 5’ splice site
3’ splice site
Exonic enhancer sequence
   
The Scheme of Spliceosome Assembly Pathway

   
The Structure of Group I and Group II Introns

Group I Group II

   
Splicing by Two Transesterification Reactions

   
Essence of Group I Introns

Guanidine binding pocket Internal Guide Sequence (IGS)

Mg2+

   
3’-End Formation of RNA

• Polyadenylation of mRNAs

• Histone mRNAs

• U-snRNAs

   
Similar 3’ Processing of Major Matazoan RNA Species

  Weiner. A. M., 2005,  Mol. Cell 20, 168-170.


Mammalian Pre-mRNA 3' Processing Sites

Mammalian polyA site

Mammalian replication-dependent histone pre-mRNA 3’ processing site

   
Model for Cleavage and Polyadenylation of pre-mRNAs

   
3’-End Formation of Histone mRNA

• Histone mRNA has no polyA tail.

• Histone pre-mRNA 3’-end is


cleaved to form the mature mRNA.

• Two recognition elements


Stem Loop
Zinc Finger Protein
HDE - histone downstream element

Stem Loop
• The cleavage reaction requires U7 snRNP Binding Protein

and other proteins.

    Dominski, Z. et al., 2005, Cell 123, 37-48.


rRNA Processing

• Processing of pre-rRNA

• Modification

   
EM of Pre-rRNA Transcription Units from
Nucleolus of a frog oocyte

   
Processing of Pre-rRNA and Assembly of Ribosomes
in Higher Eukaryotes

   
Maturation of the Ribosome

   
Decatur, W. A. and Fournier,M. J., 2003, J. Biol. Chem. 278. 695-698.
Modification of rRNA Mediated by snoRNPs

Decatur, W. A. and Fournier,M. J., 2003, J. Biol. Chem. 278. 695-698.

   
Kiss, T., 2002, Cell 109, 145-148.
RNA Editing

• Initially named in the mid-1980s to describe a novel


phenomenon in which uridine residues were inserted
and deleted from mitochondrial RNAs of kinetoplastid
protozoa.

• Currently used to describe cellular processes that result


in the modification of RNA sequences differing from that
designated by their DNA (or RNA) templates.

   
Comparison of the T. brucei COIII mRNA with the
Corresponding Mitochondrial DNA Sequence

Nature 334, 469-470 (1988).