RNA splicing and processing (lecture 9)

Question 1

percentage of protein coding genes?

percentage of transcribed genes?

what are they then?

Answer 1

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

Question 2

processing the tRNA?

Answer 2

RNaseP (a catalytic RNA) cuts off the 5’ end of the primary transcript tRNA

addition of CAA to 3’ end

base modification

Question 3

typical processing of RNA?

Answer 3

Size reduction: splicing

modification of nucleotides:

• 5‘ end: N7-methylguanosine cap (5’ cap)
• N6-methyladenosine (m6A) (3’ polyA)

Question 4

snoRNA

Answer 4

snoRNA (small nucleolar RNAs) involved in:

- cleavage, methylations, pseudouridines

Question 5

how is mRNA stability regulated?

Answer 5

mediated by N6-methyladenosine (m6A)

Question 6

key hallmarks of eukaryotic cells

Answer 6

• Nucleus-cytosol compartmentalization

- Split genes (protein-encoding genes) (Exons and Introns)

Question 7

what happens to eukaryotic transcripts?

Answer 7

• Capping at 5’
• Polyadenylation at 3’
• Splicing

all before export from the nucleus into cytosol

Question 8

pre mRNA vs mRNA

Answer 8

pre-mRNA contains Introns

after splicing (removal of introns): mRNA

Question 9

what’s the 5’ cap?

what is its function?

Answer 9

7-methylguanylate cap
by Guanylyl- transferase and 7-Methly- transferase

post transcriptionally

functions:

• mRNA stability
• splicing
• nuclear export
• translation

Question 10

what’s the 3’ polyA?

what is its function?

Answer 10

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

Question 11

what is the CTD?

Answer 11

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.

Question 12

which mRNAs are not polyadenylated?

Answer 12

histone mRNAs

Question 13

how are the splice sites characterized?

Answer 13

=splice junctions or exon-intron boundaries

characterized by short consensus sequences

GT – AG rule of intron ends

Question 14

spliceosome

Answer 14

U snRNPs

Question 15

how is splicing catalyzed?

Answer 15

RNA catalysed reaction

Mg catalysed reaction

Question 16

which type of reaction is splicing?

steps?

Answer 16

Two transesterifications reactions

involving a lariat structure

Question 17

number of introns per gene varies between organisms

Answer 17

tendency:

• > multicellular species with long generation time: intron-dense
• > unicellular species with short generation time: intron-sparse

Question 18

Potential evolutionary forces underlying intron distributions

Answer 18

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

Question 19

When and why did introns arise?

Answer 19

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

Question 20

number of genes in drosophila, c elegans and humans?

Answer 20

d: 14.000
c. e. 19.000
human: 20.000 - 25.000

alternative splicing expands the coding capacity of genomes!

Question 21

splicing and physiology/development?

Answer 21

40-70% of human genes: alternative splicing
-> influences Physiology and development

Alternative splicing can result in protein forms with different functions, different localisation etc.

Question 22

how is splicing regulated?

Answer 22

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)

Question 23

splicing without spliceosome:

Answer 23

1.) Self-splicing introns

2.) Introns cut out by nuclease cleavage
and ligation reactions

Question 24

self-splicing introns

features?

differences?

Answer 24

• 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

Question 25

introns cut out by nuclease cleavage and ligation reactions

example?

Answer 25

in pre-tRNA splicing in Archaea and Eukaryotes

in Unfolded Protein Response (UPR) pathway (yeast, mammals)

Question 26

Trans-splicing

what is it?

in which organisms?

Answer 26

splicing between different RNA molecules

Originally discovered in Trypanosomes: all mRNAs have the same 5‘-end drived from trans-splicing (mRNAs derived from polycistronic transcripts)

Nematodes (C. elegans), Trematodes e.g. C.elegans: trans-splicing (70%) and normal cis-splicing polycistronic and monocistronic mRNAs

Question 27

polycistronic transcripts

Answer 27

acterial operons are polycistronic transcripts that are able to produce multiple proteins from one mRNA transcript.

Question 28

what’s SL RNA?

Answer 28

(spliced leader RNA): forms a snRNP
-> trans-splicing and self splicing

The SL RNA adds a leader to facilitate translation -> Processing of polycistronic transcripts

(trans-splicing as a equivalent to mRNA poceesing in eukaryotes?!)

Question 29

Splicing can be coupled to which steps in gene expression?

Answer 29

to transcription (can occur co-transcriptionally)

… to nuclear mRNA export

to mRNA quality control (nonsense-mediated decay, NMD)

… to translation