RNA Processing and Translation Flashcards
Cental Dogma Steps
(in depth)
RNA Processing outline
largest known human gene
Human dystrophin
over 2.4 million bases
Requires ~16 hours to transcribe and is costranscriptionally spliced on the RBP1 platform
RBP1’s role in transcription
Part of the RNA Pol II complex. Scaffold which contains a long C-terminal domain where splicing and processing enzymes dock.
Also associates with pRb
pRb recruitment to a promoter. . .
blocks the assembly of pre-initiation complexes
RNA Processing CTD Steps
During the transition to elongation, the RNA polymerase becomes phosphorylated at specific sites. Initially, the polymerase is phosphorylated at position 5 of the repeat sequence; this recruits proteins involved in adding the 5’ cap to the mRNA (capping factors). Once capping has occurred, the polymerase becomes phosphorylated on both the 2 position and 5 position of the repeat, which helps recruit the splicing machinery. Near the end of transcription, the polyadenylation machinery (3’ end processing proteins) is recruited to the polymerase.
5’ Capping
Functions of the cap:
Protects the 5’ end from degradation by nucleases Promotes mRNA export by binding export proteins
In cytoplasm, cap binding proteins are exchanged, and promote translation
Chemistry of splicing
Sequences Recognized in Polyadenylation
Polyadenylation
Polyadenylation occurs through two steps: cleavage, followed by PolyA addition.
Poly A tails can be quite long, approximately 250 bases. Like the 5’ cap, the polyA tail also helps stabilize the mRNA (protects it from degradation by nucleases) and acts as a binding site for proteins that are involved in translation.
Sequences Required for Splicing
Mutations at these sites can interrupt the process of splicing, generally leading to skipping of the exon next to which they occur. The branch point adenine is important for the mechanism of splicing.
pre-mRNA
the name for the mRNA prior to completion of all of the processing steps
remains in the nucleus until it is processed
Splice Mechanism
Ribonucleoprotein particles called snRNPs (pronounced “snurps”) recognize the sequences in the pre-mRNA and assemble together in a structure called the spliceosome, which catalyzes splicing. Each snRNP is composed of an snRNA (called U1, U2, U4, U5, U6) plus associated proteins. They are like the ribosome in that they are ribonucleoprotein particles that catalyze a reaction.
U1 snRNP recognizes the 5’ splice site, and U2 snRNPs recognizes the branchpoint and 3’ splice site.
Next, the U4/U5/U6 snRNPs join in, and then U1 and U4 are released.
The U2/U5/U6 complex caries out the splicing reactions, and then dissociates from the RNA.
Not shown here are so-called “exon-junction complexes” that load on and remain associated with the spliced mRNA; these proteins help facilitate nuclear export of the spliced mRNA.
snRNP structure
Each snRNA contains a similar sequence called an Sm motif that recruits an Sm protein-this protein is present in all snRNPs (U1, U2, U4, U5, U6). Each snRNP carries a small nuclear RNA (snRNA, U1 snRNA, U2 snRNA, etc) as well as proteins that are unique to each snRNP. The protein components of the U1 and U2 snRNPs are shown above. The snRNAs base pair with the conserved sequences in the 5’ splice site and 3’ splice site; U2 snRNA recognizes the branch point sequence to help bulge out the adenosine that will perform the first step of splicing.
Intron vs Exon definition
Intron definition was an old theory, but could not be true as exons on the end would be lost. Exon deifnition is now the accepted true model.
Exons contain specific sequences called “exonic splicing enhancers”, or ESEs, that recruit SR proteins (they are rich in Serine (S) and Arginine (R)). The SR proteins help recruit the U1 and U2 snRNPs to the proper sites at each end of the exon. Once two neighboring exons are defined, the U1 snRNP interacts with the U2 snRNP from a different exon to initiate splicing by recruitment of U4/U5/U6. The orange protein is called U2AF for U2-associated factor; it acts as a bridge between the SR protein and the U2 snRNP.
How can weakly-binding splice factors be effective in efficient, selective, high-affinity splicing?
Works because protein complexes that help define the exon are cooperative. They each bind only weakly, but their net affinity is summative. The flip side of this principle is that if a single binding sites is mutated, it can lead to failure to define the exon, and as a result the exon will be skipped in the splicing process.
Why is exon definition important?
In higher eukaryotes, exons are small and introns are very large.
Because the sequences that promote splicing are short, the introns often have sequences that look like splice sites, but it is important that these “decoy” splice sites not be used. By including the exon in the recruitment process, the proper splice sites can be selected.
What type of mutation would cause each of these splice misfunctions?
In case B, the ESE in exon 2 has been mutated.
In rare cases, sometimes loss of a sequence will lead to use of a “weaker” sequence nearby; this can lead to extension of an exon (C) or even the creation of new exons (D). In this case, imagine a mutation that creates a new binding site for an SR proteins that is located between two decoy splice sites. This pathway of de novo exon inclusion is thought to be one pathway by which new protein sequences can evolve.
Example of extensive alternative splicing
alpha-tropomyosin
There are two basic explanations for protein isoforms: 1) gene duplication and divergence; 2) alternative splicing. Thus some proteins that have different isoforms are encoded by different genes. However, most isoforms arise by alternative splicing
Types of alternative splicing
RNA Export
Many of the proteins loaded onto the pre-mRNA as it becomes a mature mRNA are important for efficient export from the nucleus. These include the EJC (exon junction complex) proteins that are loaded on during splicing, as well as SR proteins that bind to exons. Proteins that bind to the cap are also important for nuclear export; these are exchanged for translation initiation factors in the cytoplasm.
“Pioneer” Round Translation
The first time the ribosome encounters the mRNA is called the “pioneer” round of translation, and is a bit different from subsequent rounds of translation (bulk translation). During this first round of translation, the ribosome has to “bump off” any other proteins bound to the mRNA, including the exon-junction complex proteins that remain on the mRNA (labeled EJC core) above. These complex are left as marks on the mRNA at each exon-exon junction (they essentially mark where the splice sites occur).
Overall translation error rate
~1/10,000 amino acids
