BISC 320 Chapter 10 RNA PROCESSING

precursor RNAs (pre-RNAs)

• need to be modified to make the functional mature RNA

  • types of RNA processing

    • cleavage

    • splicing

    • 5’ capping

    • polyadenylation

    • editing

mature RNA

• processed RNA ready to perform cellular function

• benefits of RNA processing

  • contribution to regulation of gene activity

  • diversity → alternative splicing (by removal of different combinations of introns)

  • quality control → defective mRNAs are detected and degraded (important for translation)

ribonuceloproteins (RNPs)

• ribosome that is like a protein

• processing complex that contains both protein and RNA

ribozymes

• RNA in RNPs that are structural or can have catalytic activities

• self splicing intron

guide RNAs

• base pair with pre-RNA and guide the RNP to correct place for processing

Processing of tRNA and rRNAs

• transcripts made as long precursors that must be processed

• encoding several RNAs in one precursor means similar amounts of each RNA are made

ribonucleases

• enzymes that cleave (degrade or process) DNA

• shorter, functional shapes

• exo and endonuclease

endonucleases

• cleave the RNA within the strand

• can be sequence specific

  • some are specific for dsRNA and ssRNA

• excision of bacterial rRNA is performed by endonuclease RNase III which recognizes dsRNA

  • binds stem-loop structures in pre-RNAs and cleaves dsRNAs

• 5’ trimming of tRNAs is done by RNase P

  • RNAase P enzymes have an RNA component as well as protein (RNP)

  • RNA component of RNAse P enzyme is critical for activity and likely contains the catalytic core of the enzyme

Addition of CCA to tRNA 3’ ends

• CCA seq at the 3’ ends of tRNAs is the attachment site for the amino acid

  • not often encoded by the genome

• CCA adding enzyme catalyzes nucleotide addition

• CCA nucleotides added sequentially

• Nucleotide binding pocket confirmation changes to control whether C or A is added

exonucleases

• remove nucleotides from the end of a transcript

• act on single strand, not sequence specific

• have a preferred direction

nucleotides of tRNA and rRNA are modified

• often chemically modified after transcription

  • help folding/stability

  • increase specificity of interactions with these molecules

• ribose 2’-O-methylation

  • CH3 added to 2’

• pseudouridylation

  • double bonded shifted and Nitrogen moved

• many rRNA mods found in regions important for ribosome function

small nucleolar RNAs (snoRNAs)

• guide RNA modification

• eukaryotes use to guide enzymes to the correct ssite

• associate with a complex of proteints to maek snoRNP

• 2’O methylation (rRNA gets made)

• found in the nucleolus and are 60-300 bp long

• base-pairs with specific regions of RNAs to direct enzyme to those positions

snoRNPs

• complex of snoRNAs and proteins

• pseudouridylation (basepaired with rRNA)

5’ capping

• eukaryotic mRNAs are capped at the 5’ end

  • protect against exonuclease digestion

• modified during transcription

• protect mRNAs from nuclease degradation and essential for translation

• added in three stages

  • RNA 5’ triphosphatase catalyzes removal of a phosphate from the 5’ end

  • a guanosine monophosphate (GMP) is attached to the end in a 5’ - 5’ triphosphate linkage

  • the guanine is methylated

(bacteria have 5’ triphosphate to be more stable)

polyadenosine tail (poly-A tail)

• the 3’ end of all eukaryotic mRNAs (except histone) have a polyadenosine, or poly(A) tail

• poly A tail protects mRNAs from degradation and is essential for translation

• after cleavage, ~200 adenosines are added by poly(A) polymerase

polyadenylation site

• mRNAs have polyadenylation sites where pre-mRNAs are cleaved (between AAUAA and U- or GU- rich region) and the poly(A) tail added at CA

3’ untranslated region (3’ UTR)

• sequence between stop codon and the polyadenylation site is the 3’UTR

• multiple poly A sites found in some mRNAs, affect translation or mRNA stablility by including or excluding regulatory sequences

  • (bacteria) attenuator, riboswitches termination of transcription

  • stem loops at 3’ ends (intrinsic terminator) and 5’ ends contribute to stability, protect against 3’ to 5’ exonuclease activity

CTD domain with 5’ capping and 3’ polyadenylation

CTD in Pol II mediates mRNA processing

• recruits capping enzyme by phosphorylated CTD

• additional phosphorylation allows splicing machinery

• recruitment of 3’ end processing complex

• spliced mRNA is cleaved and polyadenylated

once RNA processing complete, RNA is transported from nucleus to cytoplasm for translation

RNA splicing

• the removal of introns and joining of exons

  • intron is first deteached from exon 1, reacts with exon 2

alternative splicing → differential removal of introns gives different transcripts from the same gene

introns

• noncoding RNA sequences removed during splicing

• self splicing vs. excision by protein or RNPs)

• most eukaryotic introns removed by an RNP complex called spliceosome

• sometimes contain other genes (miRNAs and snoRNAs → modifies rRNA)

exons

• coding sequences retained in mature mRNA

spliceosome

• RNP complex that removes eukaryotic introns

• eukaryotic splicing mediated by spliceosome

  • made of several snRNPs

  • 100-300 nucleotide snRNA + proteins

  • not self splicing, similar to group II introns

• 3 base pairing interactions

  • snRNA to mRNA

  • snRNA to itself (hairpins)

  • snRNA to snRNA

• the rest of the snRNPs bind displacing U2AF

• first transesterfication occurs to form the lariat

• second occurs to release lariat and form spliced mRNA

transesterfications

• a single phosphodiester bond is broken and replaced by a phosphodiester bond of similar energy

• reaction doesnt require ATP

• 3’ end of intron one attacks 5’ end of exon 2

self-splicing

RNA catalyzes its own intron removal

• group 1 introns

group I introns

• found in bacteria, viruses, lower eukaryotes and plants

• ~120-450 nucleotides long

• many self-splice (excise themselves from primary transcript)

• transesterfication:

  • free g attacks 5’ end of intron

  • the released end of exon 1 attacks intron-exon2 junction

  • splice sites are defined by the three dimensional structure of intron

group II introns

• bacteria and in organellar genes of plants and fungi

• ~400-100 nucleotides long

• some are self splicing, others require cellular protiens

• 2’ OH of specific adenosine attacks the exon1-intron junction to form a lariat structure

• released exon 1 can then attack 5’ end of exon 2

• 3D structure of intron is critical for splicing

lariat

• created in group II introns

• looped intron structure formed during splicing (AGU)

small nuclear RNA (snRNA)

• combine with proteins to make up snRNPs in the spliceosomes

small nuclear ribonuclear proteins (snRNPs)

• what the spliceosome is made of (snRNA and protein)

• snRNAs form bp with pre-mRNA and work as the recognition part of the snRNP

5’ and 3’ splice sites

• spice sites are defined by short sequence motifs:

  • 5’ → GU

    • recognized by U1 snRNP

  • 3’ → AG

    • recognized by U2AF (non snRNP factor)

branch point nucleotide

adenosine where lariat will be formed

• recognized by BBP branch point binding protein (non-snRNP factor)

• U2 snRNP replaces BBP

polypyrimidine tract

right before the 3’ splice site, also recognized by U2AF

exon junction complex (EJC)

• protein complex deposited at exon-exon junctions

alternative splicing

• where different combinations of exons are used to yield more than one mature mRNA

• most exons are consecutive, but some are regulated

• can also use alternative 5’ or 3’ splice sites

• alternative TSS and polyadenlyation sites

• gene diversity

cryptic splice sites

hidden splice sites activated by mutation or error

exon definition/intron definition

mechanisms by which splice sites are recognized

intronic and exonic splicing enhancer sequences (ISE/ESE)

sequences that promote splice site recognition

intronic and exonic splicing silencer sequences (ISS, ESS)

sequences that repress splice site usage

RNA editing

• further enhances the range of molecules that can be produced

• specific nucleotide can be modified to other bases (common)

  • deamination of adenosine to inosine

  • deamination of cytidine to uridine (found in plant)

    • results in formation of stop codon only in intestine resulting in a shorter version of the protein with a unique function

• insertions or deletions can be one or two nucleotides, or can be more extensive (uncommon)

• APOB expressed in both liver and intestine

ADARs

• adenosine deaminase that acts on RNA

• often act on dsRNA (disrupts AU bp)

• inosine behaves as guanosine (translation and secondary structure)

• can affect splicing

deadenylation

shortening or removal of poly A tail causes decreased stability

northern blot

• how specific RNA sequences can be identified

• RNA is separated by size on gel, transferred from gel to membrane

• membrane is incubated with single-strand labeled probe with RNA of interest

• can detect RNA size

cDNA

DNA synthesized from an RNA template

reverse transcriptase

enzyme that synthesizes DNA from RNA

RNA degradation

bacteria

• 5’ triphosphate inhibits degradation → conversion to monophosphate by pyrophosphate hydrolase can stimulate degradation

• endonuclease begins degradation process

• degraded by 3’ to 5; exonuckease

• stem loop structures block accessibly to exonuclease → addition of poly A tail tracts help degradation as they are unstructured

eukaryotes

• usually involves series of exonucleolytic digestions

• 3’ poly A tail blocks RNA degradation → shortening of poly A tail by adenylation

• degrafation can be catalyzed in 3’ to 5’ by exonuclease

• decapping enzymes remove 5’ cap allowing 5’ to 3’ exonuclease