RNA_processing_11-06

• In cis-splicing, U1 snRNA base pairs with the 5’ splice site, whereas in trans-splicing the 5’ splice site is on the SL snRNP

• Intra- makes duplications within a gene
• Inter-connects between genes

• trans

• cis or trans

• The two most studied examples of trans-splicing between coding exons is that in mod/mdg4 and lola mRNAs.
• lola (longitudinal lacking) is a transcription factor that regulates axon guidance)

• SMaRT technology!
• can be applied to replace a 5′-, a 3′-, or an internal gene portion (=> 5′-trans-splicing, 3′-trans-splicing, or internal exon replacement)

Spliceosome-mediated RNA trans-splicing

Splicing is connected to mRNA export, stability, localization, and translatability

intron-less RNA derived from cDNA

copy/complimentary DNA

The difference between cRNA and in-vivo spliced RNA is the presence of some sort of a “memory tag” on the spliced RNA

Exon Junction Complex (EJC)

• provides a 'memory tag' for spliced RNA
• a protein complex that assembles during splicing at exon-exon junctions and assists in RNA transport, localization, translation, and degradation

• The EJC assembles on mRNA during splicing 20-24 nt upstream of exon-exon junctions

• The EJC core physically interacts with proteins involved in RNA transport, localization, translation and stability

• The EJC is transported with the mature mRNA to the cytoplasm and remains associated with the mRNP until the mRNA is translated

• The core EJC complex consists of four proteins, MLN51/BTZ, Magoh, Y14, and eIF4AIII, that form a dynamic binding platform for a variety of peripheral factors involved in mRNA metabolism
• mRNA binding is mediated by a DEAD-box RNA helicase eIF4AIII domain 2
• Inhibition of eIF4AIII ATPase activity by Magoh-Y14 forms the mechanistic basis for the long-term stability of the complex

The core of the EJC remains unchanged during mRNP maturation, but peripheral proteins associate and dissociate throughout the mRNP's journey in the cell

Nonsense-mediated mRNA decay (NMD) is a translation-dependent surveillance process that recognizes and degrades mRNAs containing a premature translation termination codon (PTC)

• nonsense-mediated mRNA decay (NMD)
• NMD prevents the synthesis of aberrant and potentially deleterious truncated proteins
• ~30% of all known human disease-associated mutations generate a nonsense mRNA
• In mammals, a stop codon is considered as premature when at least one EJC is present downstream of that stop codon

• During pioneer round of translation, EJCs are usually displaced by the scanning ribosome
• If translation terminates prematurely, the ribosome never reaches and fails to strip the final EJC

• During pioneer round of translation, EJCs are usually displaced by the scanning ribosome
• If translation terminates prematurely, the ribosome never reaches and fails to strip the final EJC
• The EJC that remains on the mRNA recruits NMD factors called UPF1,2,3 and SMG1,5,6,7
• Assembly of the NMD factors on aberrant RNA results in recruitment of decapping enzymes (DCP), as well as of endo- and exonucleases that carry out degradation of that mRNA

• Nonsense mediated Decay (NMD) in mammals (pt1)
• A translation termination event at a PTC upstream of an EJC leads to the formation of the SURF complex, which consists of SMG1 kinase, UPF1 helicase, and the ribosome release factors eRF1 and eRF3
• SURF interacts with UPF2, UPF3 and additional EJC proteins

• Nonsense mediated Decay (NMD) in mammals (pt2)
• The interaction of the SURF complex with the EJC results in the formation of the DECID (decay-inducing complex) which triggers UPF1 phosphorylation by SMG1 and the dissociation of eRF1 and eRF3 and the ribosome
• UPF1 phosphorylation leads to the recruitment of SMG5, SMG7 and SMG6 proteins and the mRNA is degraded by SMG6-mediated endonucleolytic cleavage and by exonucleolytic decay

• In plants, fungi, and insects the NMD pathway is independent of splicing and the exon junction complex
• In these species, binding of specific RNA-binding proteins to their cognate RNA elements in the 3’UTR (yeast), the strength of a signal from PABP (flies), and/or the length and structure of the 3’ UTR (plants) are the factors determining whether an RNA is recognized as a target of NMD

3–10% of all transcripts are believed to be subject to NMD

NMD reduces genomic noise by targeting transcripts originating from non-functional pseudogenes, transposable elements, and opposite (antisense) strands of coding regions

NMD

• NMD reduces genomic noise by targeting transcripts originating from non-functional pseudogenes, transposable elements, and opposite (antisense) strands of coding regions
• NMD also detects incompletely spliced mRNAs that escaped from nuclear retention
• Global expression analyses revealed that some “normal” genes, such as those involved in the regulation of chromosome structure and behavior (e.g., telomere replication and maintenance, chromatin silencing, recombination and repair) are also regulated by NMD

degrades mRNA that lack an in-frame stop codon

• Non-stop transcripts are generated by premature transcription termination and polyadenylation at a “cryptic” site
• Ribosome continues translating into poly(A), striping PABP, and eventually stalls
• PABP-deprived mRNA gets degraded by the exoribonucleases: by XRN1 upon de-capping or by Ski7-recruited exosome

5-10% of all mRNAs!

• Ski7-Superkiller protein 7, has sequence similarity to eRF3
• recruits an exosome and degrades PABP-deprived mRNA

• degrades mRNA with stalled ribosomes
• Ribosomes stall upon encountering a rare codon or an extensive 2o structure (stem loop) on the RNA
• Dom34 and Hbs1 (~eRF1 and eRF3) bind to the the ribosome, cleave mRNA, release arrested ribosome, and send nascent peptide to degradation

• No-go decay (NGD)
• found in yeast

Non-stop decay (NSD)

• Ribosomal RNA
• structural and functional components of ribosomes

• rRNA genes in eukaryotes are organized in tandem array, with individual rRNA gene copies separated by nontranscribed spacer regions ranging in length from ≈2 kb in frogs to ≈30 kb in humans
• In humans, there are approximately 300–400 rDNA repeats organized in five clusters (on chromosomes 13, 14, 15, 21 and 22)

• in the nucleolus
• Both transcription and processing of eukaryotic rRNAs and assembly of rRNA into ribosomes takes place in the nucleolus

Three different rRNA molecules, 18S, 5.8S, and 28S (always in that order!) in eukaryotes, are contained in a single transcript and need to be individually cut out

RNAP I

RNAP III

small nucleolar RNPs (snoRNPs) that consist of  snoRNAs (e.g., E1, E2, E3, U3, U14) and a number of associated proteins

box H/ACA snoRNA

C/D snoRNA

• Peptidyl transferase center (PTC)
• the A, P, and E sites where tRNA and mRNA bind
• the polypeptide exit tunnel
• sites of subunit–subunit interaction

rRNA modifications are thought to be necessary for proper rRNA folding and/or association with chaperone proteins that aid in folding and, therefore, for ribosome assembly

PTC is the catalytic center of the large subunit of the ribosome