Lecture 14: RNA Processing II

rNA Processing II Side Notes: Splicing can yield more than one mature RNA • The Majority of genes in more complex eukaryotes undergo alternative splicing where different combinations of exons are used • Most exons are constitutive (must be included), but some are regulated exons (can be included or not) • In addition, alternative splice sites can be used, at the 5’ end of the 3’ end • Alternative transcriptional starts can be used and different termination sites Exon definition model of splice site recognition • The spliceosome must be able to recognize only true splice sites. There are two models for how this works; exon definition and intron definition • Exon definition is thought to occur in mammals: the 5’ and 3’ ends of an exon are brought together by interactions between U1 and U2 complex. Mutations at a 5’ splice sir would result in exon exclusion Intron definition model of splice site recognition • Intron definition: in this model, introns are defined by interactions between 5’ and 3’ bound factors. Mutations at a 5’ splice site would result in intron inclusion • When exons are “defined”, splicing intermediates must be reorganized, but this is not needed when introns are “defined” Splice junction recognition • In both exon definition and intron definition models, the final splicing complex must be one in which the 5’ and 3’ complexes interact with one another across the intron • The 5’ and 3’ splice sites are marked as they are transcribed (i.e., cotranscriptionally), facilitated by RNA polymerase and splicing components • The exon is typically marked with SR proteins and the introns bound with hnRNPs, which masks cryptic splice sites Additional sequences affect spliceosome function • RNA sequence elements that positively affect splicing are called splicing enhancers, and can be intronic splicing enhancer sequences (ISE) or exotic splicing enhancer sequences (ESE) • Silencer sequences have the opposite effect and can also be intronic or exonic (intronic splicing silencer sequences; ISS, and exonic splicing silencer sequences; ESS) • An example of a silencer protein is polypyrimidine tract binding protein (PTB), which binds to intron elements and silences neighboring weak introns Alternative splicing of the gene encoding SRC tyrosine Kinase • Polypyrimidine tract binding protein (PTB) is an intronic splicing silencer protein • In non-neuronal cells PTB binds to either side of the N1 exon, and it is repressed from joining the final mature mRNA • In neuronal cells PTBP2 binds to the same elements instead of PTB. PTBP2 does not repress inclusion of N1, and an enhanced sequence is activated that stimulates N1 splicing, so N1 is included Nucleotides can be modified, inserted, or deleted in mRNAs • There are three main common RNA Edits: ◦deamination of adenosine to inosine (the most common edit in more complex eukaryotes). Inosine is interpreted as guanosine, so changes in the coding region can final protein sequence rNA Processing II Side Notes: ◦Deamination of cytidine to uridine, which has been found to date mainly, but not exclusively, in plant mitochondrial and chloroplast mRNAs ◦Methylation of adenosine, revealed by deep sequencing • Nucleotide conversions like these are widespread throughout life • Uridine insertions and deletions have only been found so far in mitochondrial genders of single-called eukaryotes like trypanosomes Trypanosomes have an extreme form of mRNA editing • The Trypanosoma brucei NADH dehydrogenase 7 gene undergoes extensive editing • For such modified genes, it is difficult to predict the protein sequence from the DNA sequence alone Cytidine to uridine deamination can cause alternate stop codons • Cytidine to uridine deamination is observed in the mRNA that makes human apoliprotein B • The deamination of a particular cytidine results in formation of a stop codon, resulting in a shorter version of the protein - this occurs in the small intestine, and is needed for lipid absorption from food • The long version of the protein is made in the liver, and is involved in cholesterol transport Adenosine methylation is reversible • Reversible methylation of adenosine is mediate by ‘writer’ and ‘eraser’ proteins in the nucleus • Methylated adenosines are read by ‘reader’ proteins in the cytoplasm • Binding of ‘reader’ proteins to methylated adenosines in 5’ UTRs may be involved in regulation of translation RNA degradation • RNAs need to be degraded at some point, removing RNAs that are no longer needed and recycling the nucleotides • Some RNAs, like rRNAs, are needed a lot, and are fairly stable. Others, like some mRNAs, are only required for short periods of time and so are rapidly degraded • RNA stability is described as RNA half-life: the time in which the amount of RNA is reduced by half. RNA half-lives range from <1 minute to an hour in E. coli and ~20 minutes to >24 hours in vertebrates Endogenous RNA degradation in bacteria • In bacterial mRNAs, degradation is often initiated by an endonuclease, usually RNase E • The products of the first digestion are then degraded by 3’ to 5’ exonucleases • RNase E is part of the degradosome complex. The complex also has an RNA helicase and a 3’ to 5’ exonuclease • A 5’ triphosphate inhibits RNase E activity - this is converted to a monophosphate by RppH • Stem-loop structures inhibit exonucleases. 3’ poly(A) tracts help degradation as they are unstructured • 3’ poly(A) tails hinder rather than aid degradation in eukaryotes - the first step in degradation is often tail shortening by a deadenylase • After this, decapping enzymes remove the 5’ cap and 5’ to 3’ exonucleases degrade the RNA, or the exosome catalyzes CCR4 exonuclease activity in the 3’ to 5’ direction rNA Processing II Side Notes: The Poly(A) tail is a determinant of mRNA degradation • AU-rich elements (AREs) are important for mRNA stability • AREs are found in the 3’ UTRs of some mRNAs • ARE presence directs poly(A) removal and increases mRNA instability • c-fos in vertebrates encodes an ARE-containing short-lived transcription factor that promotes cellular growth • Some tumor-causing viruses express v-fos, which does not have the ARE • The v-fos transcript is not degraded properly and this leads to excessive cellular growth RNA degradation can be regulated by signals • Mammalian cells respond to iron levels. Iron concentration is partly regulated by the transferrin receptor, which imports transferrin-bound iron into the cell • When iron levels in the cell are high, the mRNAs encoding the receptor are degraded, as there is no need to import more iron • When cellular iron is low, the receptor mRNA is protected from degradation by the regulatory proteins (IRP1 and IRP2) that bind to stem-loop iron response elements (IREs) Foreign RNAs are degraded by alternative pathways • Some types of molecules can be harmful to the cell - such as foreign RNA or RNA from viruses or defective endogenous RNAs • Foreign nucleic acids are removed by RNA interference (RNAi) in eukaryotes and CRISPR interference in bacteria • In RNAi, foreign dsRNAs are chopped into 20-30 nt fragments • The short fragments are loaded onto Argonaute proteins that have nuclease domains • The RNA fragment guides the protein to the foreign RNA and the nuclease degrades it • In bacteria, clustered regularly interspaced palindromic repeats (CRISPR) are involved in degradation of foreign DNA • The foreign DNA is integrated into the CRISPR locus • The locus is then transcribed and processed into short fragments • As in RNAi, the short fragments act as guide molecules to direct the degradation of the target DNA or RNA Defective mRNAs removed by specific decay mechanisms • Defective endogenous RNAs are removed by specific decay mechanisms • mRNAs can have additional or absent stop codons, or other problematic coding sequences that stall translation • In eukaryotes, ribosomes on defective RNAs are marked by interaction with proteins such as the EJC, which recruit RNases to degrade the RNA • This is part of a process known as nonsense-mediated decay (NMD) • In bacteria, stalled ribosomes are recognized by a complex containing tmRNA (as RNA that acts both as a tRNA and as an mRNA) • tmRNA binding allows the ribosome to finish translation using the tmRNA as template • tmRNA also aids recruitment of RNases RNA is bound by RNA recognition motif domains • RNA-binding proteins have specific RNA-binding motifs - these are different from DNA-binding motifs because of the structural differences between RNA and DNA • The most common is the RNA-recognition motif (RRM) rNA Processing II Side Notes: • This has alpha helices and four beta sheets in a sandwich - this is very adaptable and can bind RNAs that have different structures • For example, RRM in hamster nucleolin and human PABP bind very differently structured RNAs • KH and PAZ domains also bind single-stranded RNA • KH domains have an RNA-binding groove formed by two alpha helices and one beta strand • PAZ domains are found in many RNAi proteins, like dicer • PAZ domains have a pocket formed from beta strands and an alpha helix; this pocket interacts with the single-stranded overhangs of the siRNA • The PAZ domain and binding pocket of human Argonaute 2 is shown Some RNA-binding domains recognize double-stranded RNAs • RNase III family enzymes have double-stranded RNA-binding domains • These proteins have an alpha helix and two loop regions that interact with three grooves on the double stranded RNA • The interacting grooves are minor, major and another minor Some RNAs are recognized by proteins with multiple repeated motifs • RNA-binding proteins often have more than one binding motif • The eukaryotic PUF protein family, including Pumilio 1 has repeats of a two helix motif, with conserved amino acids, each interacting with a single nucleotide • Some RNA-binding proteins need to have small regions rich in arginine and lysine (basic) • Some proteins with the zinc finger DNA-binding domains also bind to RNA