RNA splicing and processing (lecture 9) Flashcards
(29 cards)
percentage of protein coding genes?
percentage of transcribed genes?
what are they then?
2 % coding
60-70 % transcribed
mRNA, rRNA
Short RNAs: e.g. tRNA, snRNA, snoRNA, miRNA, piRNAs, endo-siRNAs, unannotated short RNAs
Long non-coding RNAs (lnc RNA; >200bp): e.g. XIST,
enhancer RNAs
processing the tRNA?
RNaseP (a catalytic RNA) cuts off the 5’ end of the primary transcript tRNA
addition of CAA to 3’ end
base modification
typical processing of RNA?
Size reduction: splicing
modification of nucleotides:
- 5‘ end: N7-methylguanosine cap (5’ cap)
- N6-methyladenosine (m6A) (3’ polyA)
snoRNA
snoRNA (small nucleolar RNAs) involved in:
- cleavage, methylations, pseudouridines
how is mRNA stability regulated?
mediated by N6-methyladenosine (m6A)
key hallmarks of eukaryotic cells
- Nucleus-cytosol compartmentalization
- Split genes (protein-encoding genes) (Exons and Introns)
what happens to eukaryotic transcripts?
- Capping at 5’
- Polyadenylation at 3’
- Splicing
all before export from the nucleus into cytosol
pre mRNA vs mRNA
pre-mRNA contains Introns
after splicing (removal of introns): mRNA
what’s the 5’ cap?
what is its function?
7-methylguanylate cap
by Guanylyl- transferase and 7-Methly- transferase
post transcriptionally
functions:
- mRNA stability
- splicing
- nuclear export
- translation
what’s the 3’ polyA?
what is its function?
The 3‘ ends of eukaryotic mRNAs are generated by cleavage and polyadenylation
- endonuclease cleaves at poly-Adenylation site
- poly(A) polymerase adds 200 A residues
- poly(A) binding proteins (PBP)
function:
- mRNA stability
- translation
- transcriptional termination
what is the CTD?
RNA pol II associates with a large number of enzymes and protein/RNA-binding factors through its C-terminal domain (CTD) that consists of tandem repeats of the heptapeptide consensus Y(1)S(2)P(3) T(4)S(5)P(6)S(7). The CTD is posttranslationally modified, yielding specific patterns (often called the CTD code) that are recognized by appropriate factors in coordination with the transcription cycle.
coordinates transcription and RNA processing
Serine phosphorylations are currently the best characterized elements of the CTD code; however, the roles of the proline isomerization and other modifications of the CTD remain poorly understood. The dynamic remodeling of the CTD modifications by kinases, phosphatases, isomerases, and other enzymes introduce changes in the CTD structure and dynamics. These changes serve as structural switches that spatially and temporally regulate the binding of processing factors. Recent structural studies of the CTD bound to various proteins have revealed the basic rules that govern the recognition of these switches and shed light on the roles of these protein factors in the assemblies of the processing machineries.
which mRNAs are not polyadenylated?
histone mRNAs
how are the splice sites characterized?
=splice junctions or exon-intron boundaries
characterized by short consensus sequences
GT – AG rule of intron ends
spliceosome
U snRNPs
how is splicing catalyzed?
RNA catalysed reaction
Mg catalysed reaction
which type of reaction is splicing?
steps?
Two transesterifications reactions
involving a lariat structure
number of introns per gene varies between organisms
tendency:
- > multicellular species with long generation time: intron-dense
- > unicellular species with short generation time: intron-sparse
Potential evolutionary forces underlying intron distributions
Classical interpretation:
-introns and non-essential DNA are disfavoured by short genome replication time
More recent suggestions:
- Population size as driving variable: Introns impose weak selective disadvantage (special sequence requirements for splicing) -> Slightly deleterious alleles are more likely to become fixed in small populations (intron-rich)]
- Differences in recombination rate determine intron loss: Intron loss (via gene conversion by RT-mRNA) depends on recombinantion; number
When and why did introns arise?
Introns-early model: numerous already in ancestors of eukaryotes and prokaryotes
- Intron-late model: recent insertion after divergence of eukaryotes and prokaryotes
Why?:
- “Exon shuffling“: recombination within introns facilitated evolution of early (multidomain) genes (-> exons encode discrete elements of protein structure)
- Increased intra-genic recombination by introns (elimination of mutations, may increase fitness)
- Alternative splicing: expanding coding capacity of genomes
number of genes in drosophila, c elegans and humans?
d: 14.000
c. e. 19.000
human: 20.000 - 25.000
alternative splicing expands the coding capacity of genomes!
splicing and physiology/development?
40-70% of human genes: alternative splicing
-> influences Physiology and development
Alternative splicing can result in protein forms with different functions, different localisation etc.
how is splicing regulated?
splicing is regulated via exonic or intronic sequences
Can involve:
- Different ratios of ubiquitously expressed splice factors
(e. g. SR proteins , hnRNPs A/B) - Tissue or cell-type specifically expressed splice regulators (e.g. NOVA, FOX proteins)
- Signaling-induced modification of splice regulators (e.g. Sam68, TIA 1)
splicing without spliceosome:
1.) Self-splicing introns
2.) Introns cut out by nuclease cleavage
and ligation reactions
self-splicing introns
features?
differences?
- transesterification reactions cut them out
- build extensive secondary structure
- contain open reading frames
- can be mobile genetic elements
In contrast to group I introns, intron excision occurs in the absence of GTP and involves the formation of a lariat, with an A-residue branchpoint strongly resembling that found in lariats formed during splicing of nuclear pre-mRNA. It is hypothesized that pre-mRNA splicing (spliceosomal introns and the spliceosome) may have evolved from group II introns, due to the similar catalytic mechanism as well as the structural similarity
discovered in tetrahymena
