RNA transcription and processing Flashcards
What are the different functions of RNA?
Protein translation: rRNA, tRNA
Protein coding: mRNA
regulation of gene expression: miRNA, lncRNA, sncRNA, psuedogene transcripts(?), circular small RNA(?)
rRNA processing: snoRNA
RNA splicing and telomere maintenance: snRNA
What are DNA coding and template strands?
Coding: 5’ -> 3’ and same sense as mRNA
Template: antiparallel 3’ -> 5’, strand read by polymerase
What are the functions of the different eukaryotic RNA polymerases?
DNA template dependent polymerases:
- Pol I = rRNA
- Pol II = protein encoding and snRNA
- Pol III = small RNAs (rRNA, tRNA, snRNA, siRNA)
- POLRMT = mitochondrial RNA
Non-template dependent:
- Polyadenylate Pol = mRNA polyadenylation
- Poly ADP-ribose Pol (PARP) = protein modification
How are coding and noncoding sequences distributed across a chromosome?
- protein coding genes are dispersed
- rRNA genes are near telomeres
- satellite DNA (highly repetitive gene poor sequences) are near the centromere
- rRNA and tRNA genes each in tandem repeats (multiple copies) in regions between genes
What characteristics do all eukaryotic polymerases share?
- highly processive
- 5’ -> 3’
What kind of genes are found in tandem repeats?
- middle repetitive regions of DNA
- multiple copy genes like rRNA and tRNA genes
- mini-satellites = variable number tandem repeats ( > 5 bp)
- micro-satellites = dinucleotide repeats ( < 5 bp)
What are interspersed transposons?
- Middle repetitive regions of DNA coming from ancient retroviruses.
- Can be active of inactive; active ones encode RT and can jump about the genome
- SINES (short interspersed elements) = alu genes involved in development
- LINES (long interspersed elements) = L1
How are protein coding genes distributed across chromosomes?
- Gene dense, poor, and middle
- Always no protein coding genes near centromere
- VERY FEW protein coding genes on the Y chromosome
- rRNA genes only found on 5 chromosomes
Describe the steps of rRNA transcription and processing.
- Transcription
- rRNA genes transcribed from tandem repeats, including ITS (internally transcribed space) regions. - Chemical modification:
- pseudouridine
- 2’-O-methylation - Degradation and cleavage
- ITS regions degraded - Assembly
- 18S, 5.8S, and 28S rRNAs joined by 5S rRNA which is made elsewhere
Where does ribosome biosynthesis occur?
- 5 rRNA containing chromosomes coalesce in the nucleolus.
- Transcription, translation, and modification (methylation, trimming) occur in the nucleolus.
- Individual subunits are exported to the cytoplasm via nuclear pores
- Assembly occurs in cytoplasm
Describe tRNA structure.
- Stem-loop structure
- Counter clockwise 5’ -> 3’: D loop, anticodon loop, variable loop, T-psi-C loop, amino acid attachment site.
- Amino acid attachment site = 3’ ss CCA
- D = dihydrouridine
- T = ribothymidine
- psi = pseudouridine
How are tRNAs processed?
- Transcription of tRNA precursor in nucleus
- Removal of intron
- Base modification (D, T, and psi) and addition of 3’ CCA
- Export to cytoplasm through nuclear pore
Describe the upstream promoter elements that regulate RNA Pol II transcriptions
Proximal control elements:
- GC box at -100 (GGGCGG)
- CAAT box at -80 (GCCCAATCT)
Core promoter:
- TATA box at -25 (TATAA)
How is RNA Pol II transcription initiated by recruitment of basal transcription machinery?
- Basal TF (TFIID with TBP) binds TATA box
- Recruits and binds TFIIB at TATA box
- RNAP II and associated factors (TFIIF, TFIIE, TFIIH, and others) associate with the promoter
- RNAP II c-terminal tail is phosphorylated
- Transcription begins
What is the mediator complex?
- Regulates Pol II gene expression
- Contains proteins and factors that bind to upstream enhancers (regulatory sequences can be very far upstream)
- Includes chromatin-remodeling complex, histone-modifying enzyme, and other machinery needed to expose/loop DNA by unwinding and removing histones
What is the function of an enhancer? How do they work and where are they located?
- To change the activity of a promoter; promoter activity in the absence of an enhancer is by default low (basal)
- Regulatory proteins (transcription factors) bind enhancer DNA sequences
- Activators increase transcription and repressors decrease transcription
- Can be > 50kb upstream/downstream of the promoter region or even within a gene
What histone modifications regulate transcription?
- lysine methylation: transcriptional activation or repression depending on context
- lysine acetylation: transcriptional activation
- cytosine modification (5-methyl-cytosine): transcriptional repression
How does cellular metabolism affect transcriptional regulation?
- Substrates that add/remove methyl (a-KG, SAM) and acetyl (acetyl-CoA, acetate) groups to histones come from metabolism
How is tissue specific gene expression regulated if all cells share the same DNA?
Tissue specific transcription factors needed to activate promoter
What is splicing?
Removal of introns from pre-mRNA
What are hnRNPs?
- heteronuclear ribonucleoprotein particles
- pre-mRNA transcripts bind to RNPs during transcription and post-transcriptional modification, signaling that they are not yet ready for export to they cytoplasm
When are the 5’ cap and poly-A tail added to an m-RNA? When does splicing occur?
Co-transcriptionally
How are Pol II RNA transcripts co-transcriptionally processed?
- Carboxyl terminal domain (CTD) or Pol II is phosphorylated, allowing for binding of capping, splicing, and polyadenylation factors
What is the polysome?
Multiple RNAP II’s on a single gene: as soon as one moves downstream another binds and begins transcription
What proteins are responsible for pre-mRNA splicing?
small nuclear riboproteins (snRNPs): U1, U2, U4, U5, U6
Where are the splice donor, splice acceptor, and branch sites located and what are their sequences?
- Splice donor = GU = 5’ end of intron
- Branch site = A + hydroxyl = within intron
- Splice acceptor = AG = 3’ end of intron
What is the mechanisms of pre-mRNA splicing?
Complex assembly: U1 snRNP binds splice donor site and U2 snRNP binds branch site. U4/U5/U6 bind pulling U1 and U2 together and forming the snRNP complex. U5 snRNP binds to splice donor and acceptor sites.
- Cleavage of intron from exon 1 at splice donor site
- Nu attack of branch site hydroxyl to splice donor results in lariat formation (covalent link)
- Cleavage of intron from exon 2 at splice acceptor site.
- Ligation of exons
What is the purpose of alternative splicing? What are some mechanisms of alternative splicing?
- Generates protein diversity from identical transcripts; usually making isoforms in different cell types
Types:
- optional exon
- optional intron
- mutually exclusive exons
- internal splice site
What is cryptic splice site utilization and how is it involved in disease?
- Mutations to an intron can create an internal splice acceptor or donor site
- Exons may be added/removed; reading frame may shift
- Protein structure will be modified
How are poly(A) tails added to the 3’ end of primary transcripts?
- A2UA3 sequence near the 3’ end signals for downstream poly(A) addition
- Cleavage 15-30 bp downstream (sloppy - not consistent)
- Poly(A) polymerase adds A tail
How can mutations cause problems with poly(A) tailing?
- Poly(A) signal sequence (A2UA3) can be created earlier in a gene leading to earlier cleavage
- Changing the length of the 3’ UTR may remove important 3’ regulatory sequences
How are processed messenger RNPs exported from the nucleus?
- Modification proteins (cap-binding protein, exon junction complex, and poly(A) binding protein) are left on the mRNA after modification as proof.
- Export through nuclear pore complex
- Exchange of 5’ cap binding protein for translation initiation factors
- if modification proteins are not present, the nonsense mediated decay proofreading pathway will be activated
Describe the transcription and processing of miRNAs.
- miRNA gene is 20-22nt that could be in a coding, noncoding, or intergenic region
- Transcription by RNA Pol II
- Stem loop structure forms
- Drosha/DGCR8 complex removes ends => pre-miRNA
- pre-miRNA exported to cytosol
- Dicer/TRBP cleaves loop
- miRNA duplex split into passenger strand and guide strand
- passenger strand degraded
- Argonaut protein of RISC complex binds guide strand
- mature miRNA binds DNA/RNA and can be used to inhibit translation
How can RNA be edited and what are the effects of editing?
edits:
- adenosine to inosin
- cytidine to uridine
effects:
- specific AA substitutions
- alternative splicing
- changes in expression levels
How can gene expression be regulated via transcriptional control?
- epigenetics: chromatin remodeling and DNA methylation
- gene rearrangement
- gene amplification
- gene deletion
- TF availability
- non-coding RNAs (miRNAs, lncRNAs)
How can gene expression be regulated post-transcriptionally?
- alternative splicing
- alternative polyadenylation
- alternative start site selection
- RNA editing
- RNA stability
- non-coding RNAs (miRNAs, lncRNAs)
- regulated nuclear export
