11-2-11 RNA Splicing and Protein Synthesis1. Processing of ribosomal and transfer RNAs

2. mRNA modification and splicing

3. Catalytic functions of RNA

4. The genetic code

5. Amino acid activation

6. Ribosome structure

7. Protein synthesis

a. initiation, elongation and termination

b. inhibition of protein synthesis

8. Secretory and membrane proteins

9. Regulation of protein synthesis

Virtually all initial transcription products are processed in eukaryotes

Eukaryotic ribosomal RNAs are generated by cleavage of a precursor molecule

Nucleolar RNA polymerase I transcribes a single 45S precursor containing 18S, 28S and 5.8S rRNAs

18S rRNA – component of small 40S subunit

28S, 5.8S rRNAs – components of large 60S subunit

The 5S component of the 60S subunit is transcribed by RNA polymerase III

Nucleotides in the pre-rRNA are extensively modified prior to cleavage

Modification of pre-rRNA bases and ribose iscatalyzed by snoRNPs (small nucleolar ribonucleoproteins) consisting of snoRNA and several proteins

Cleavage and additional modification of pre-rRNAleads to production of mature rRNAs that are assembled together with ribosomal proteins into eukaryotic ribosomes

Virtually all steps occur in the nucleolus

RNA Polymerase III transcribes Transfer RNAs that are then extensively processed

5’ nucleotides (the 5’ leader) are cleaved by RNase P

CCA, the amino acid attachment site, is added to the 3’ end by CCA adding enzyme

tRNA bases and riboses are extensively modified

Some pre-tRNAs contain introns that must be removed by splicing by endonuclease and ligase

Messenger RNAs are modified and spliced

RNA polymerase II transcription products are extensively modified

The 5’ end of pre-mRNA is modified by addition of a 5’-5’ cap consisting of 7-methylguanylate (cap 0)

Adjacent ribose residues may be methylated to form cap 1 or cap 2

5’ Caps stabilize mRNAs and enhance translation

Most pre-mRNA 3’ ends are modified by polyadenylation to create poly(A) tails

3’ nucleotides are removed from the primary transcript before addition of poly (A)

An internal AAUAAA sequence in the primary transcript is recognized by a specific endonuclease that removes downstream nt’s

Poly(A) polymerase then adds about 250 adenylate residues to the 3’ end of the transcript

Poly(A) tails stabilize the transcript and enhance translation efficiency

Introns are spliced from pre-mRNAs

Introns are precisely marked by splice sites

Introns begin with GU and end with AG

5’ splice sites are marked by the consensus sequence AGGUAAGU in vertebrates

3’ splice sites are marked by the polypyrimidine tract (10 U or C residues)

Small nuclear RNAs in spliceosomes catalyze pre-mRNA splicing

snRNAs contain fewer than 300 nucleotides and some are essential to the splicing process

snRNAs associate with specific proteins to form small nuclear ribonucleoprotein particles (snRNPs), or “snurps”

In mammals splicing is initiated by recognition of the 5’ splice site by the U1 snRNP, which contains a 6 base pair sequence that base pairs with the 5’ splice site

U1 snRNP binding initiates spliceosomeassembly

U2 snRNP then binds the “Branch site”

Preassembled U4-U5-U6 join U1-U2 tocomplete spliceosome assembly

Splicing begins when U5 interacts withthe exon sequence in the 5’ splicesite and then the 3’ exon

U6 disengages from U4 and interacts with U2 and the 5’ end of the introndisplacing U1

U2 and U6 thus form the catalytic center

U4 is an inhibitor that masks U6until the specific splice sitesare aligned

The ends of the intron are thus brought together, resulting in“transesterification”

The 5’ end of the intron is cleaved toproduce a lariat intermediatewith the first G of the intronlinked to the A in the branchregion

U5 holds the 3’ end of exon 1 nearthe 5’ end of exon 2, resultingin transesterification 2

Transesterification 2 connects exon 1 with exon 2,

generating the spliced product

U2, U5 and U6 bound to the excised lariat intron are

released to complete the splicing reaction

ATP powered RNA helicases are required to unwind

RNA helices and create the alternative base pairs

needed in splicing

Mutations that affect Pre-mRNA splicing cause disease

Mutations can be cis-acting (affecting pre-mRNA) or trans-acting (affecting splicing factors)

Cis-acting mutations cause some thalassemias

hereditary anemia caused by defective

hemoglobin synthesis

The hemoglobin gene has 3 exons and 2 introns

Cis-acting mutations can affect splice sites

Splicing mutations result in incorrectly spliced mRNA that create translation stop sites preventing formation of full length hemoglobin

Mutations affecting splicing are estimated to cause

at least 15% of all genetic diseases

Alternative splicing yields protein diversity

Different combinations of exons within the same

gene may be spliced into mature RNA to

produce distinct forms of the protein for specific tissues, developmental stages or signaling pathways

Alternative splicing is controlled by trans-acting factors that differ in different cells

Alternative splicing expands the versatility of genomes via combinatorial control

In humans two different hormones are produced from a single calcitonin-CGRP pre-mRNA

calcium andphosphatemetabolism

calcitonin-gene-related protein, a vasodialator

RNA can function as a catalyst - Ribozymes

Splicing is mainly catalyzed by RNA molecules,

with proteins playing a supporting role

RNase P has an RNA component that contributes

to cleaving nucleotides from the 5’ end of tRNA precursors

Ribosomal RNAs are catalytic during translation

Ribosomal RNA processing in Tetrahymena contains a 414 bp “self-splicing” intron