BIOL 300 - Chapter 19

do prokaryotes undergo formal translation?

no, because bacterial mRNA doesn’t have introns and is directly translated as it’s being transcribed to form a polyribosome

what is an example of why RNA needs to be processed?

unmodified uridine being modified into pseudouridine is needed to prevent inflammatory responses by the immune system - the modification is the reason behind mRNA > protein stability

what is a non-coding region called?

intron

what is a coding region called?

exon

what does the process of mRNA processing include?

capping, splicing, and addition of polyA tail (in that order)

what is the function of mRNA processing?

remove introns, increase stability, and enable export

what is 5’ capping for?

stabilizing mRNA - without it, each mRNA’s first nucleotide would have reactive triphosphate

what two steps must occur before 5’ capping happens?

1) GTP is added in reverse orientation (5’ to 5’ triphosphate linkage), as RTPase removes terminal phosphates, then GTase hydrolyzes GTP to transfer it to the now diphosphate area of pre-mRNA. Next, MTase adds a methyl to the N7 of terminal G

2) The cap must be methylated, and mRNA is monomethylated on 5’ G in nucleus

when does 5’ capping occur?

in the early stages of transcription, as it’s needed for productive elongation

how does 5’ capping occur?

RNA pol 2 stops 20-30 nucleotides upstream from start site, then its CTD is phosphorylated to create binding sites for enzymes responsible for capping

what is the function/purpose of the 5’ cap?

increases mRNA stability and protects it from exonucleases, it’s required for elongation, stimulates splicing of first intron, facilitates mRNA export, and facilitates binding to translation initiation factors

what is a common method of actively degrading mRNA?

removing the 5’ cap - used to regulate the abundance of mRNA

when are introns spliced from the primary transcript in eukaryotic mRNA processing?

during transcription, while RNA is being transcribed, but only after the 5’ cap is added

what is hnRNA?

heterogenous rNA - nuclear RNA that’s much larger than mRNA, which suggests that processing occurs before being transported into the cytoplasm

what is the mechanism of intron recognition?

splicing junctions are present (both a 5’ splice site and a 3’ splice site), and a branch site that’s upstream of the 3’ splice site

what is the 5’ splice site?

starts with GU (donor sequence)

what is the 3’ splice site?

starts with AG (acceptor sequence

what is the branch site?

a sequence that specifically contains an A between 14-45 nucleotides up stream of the 3’ splice site - the sequence isn’t really conserved in animals

how do mutations or deletions in the branch site prevent splicing?

it can alter the secondary structure of the RNA, preventing the spliceosome from properly assembling and leading to incorrect splicing outcomes

what are some mutations that could affect the ORF?

exons accidentally being altered/cut, introns being accidentally included/not properly spliced out

what two transesterification reactions must occur in splicing?

lariat formation (2’ OH of the A at the branch site attacks the 5’ splice site, which makes a lariat and linear exon with a free 3’OH), and ligation of exons (3’ OH of donor attacks 3’ splice site of acceptor and connects exon 1 and exon 2

how are introns recognized?

splice junctions are read in pairs by the spliceosome, since the 5’ site is always connected to the 3’ site that comes next

what two kinds of mutations could occur in non-coding sequences that could result in consequences?

mutations in promoter regions (leads to alteration/prevention of expression), and mutations in splicing junctions/branch site (leads to intron retention and exon loss)

what is snRNA?

small nuclear RNA

what does the spliceosome consist of?

5 snRNAs, 41 proteins in snRNPs, 70 splicing factors, and 30 other miscellaneous proteins

what is the U2 spliceosome?

(major intron) part of the larger spliceosome complex; splices 98% of RNA’s

what is the U12 spliceosome?

(minor intron) part of the larger spliceosome complex; splices 2% of RNA’s

what RNA components does the spliceosome consist of?

5 snRNA’s: U1, U2, U4, U5, U6, that all have the function of base pairing between snRNA and pre-mRNA, or between snRNAs - all also have secondary structures like hair pins

what protein components does the spliceosome consist of?

there are 5 nsRNPs that correspond to the 5 snRNA, and each snRNA has ~12 snRNPs, each of which are named after the snRNA they associate with, along with 70 protein factors involved in spliceosome assembly, and 30 other proteins associated with the spliceosome that are involved in other stages of gene expression

what steps are involved in the assembly of the spliceosome complex?

donor site is recognized by U1 snRNP (U1 5’ bp is needed but also protein factors like SR), branch site is recognized by branch point binding proteins, acceptor site is then recognized by U2AF, then exon/intron boundary is bridged together by SR binding to both U1 and U2AF, which forms the E complex

what is the role of the E complex?

bring donor site, branch point, and acceptor stie together - if it’s not formed, the strand won’t commit to splicing.

what is the snRNA activity in spliceosome assembly?

1)U1 will bp with the 5’ splice site

2)U2 will bp with branch site and consume ATP

3) U4/6 and U5 trimer will join

4)As the spliceosome activates, U1 and U4 are released

5)First transesterification happens and 5’ is cleaved

6)Second transesterification happens, and 3’ end is cleave and exons are joined together

Is a specific sequence or complementary base pairing essential for splicing?

complementary base pairing

what occurs if snRNA is mutated?

the entire genome won’t be spliced correctly - a death sentence

what is alternative splicing?

one gene can produce multiple mRNA/proteins based on how it’s spliced - over 90% of mammalian pre-mRNA is alternatively spliced

what does alternative splicing result in?

encoding proteins with diff. cellular localizations, proteins with diff activities, and RNA/proteins w diff stabilities

how does RNA pol 2 terminate transcription?

termination occurs up to 1.5kb downstream from the 3’ end of the mRNA, and a cleavage signal is present to signal to cut off the RNA after that point - it’s then followed by polyadenylation

what is the cleavage signal for the end of pre-mRNA?

AAUAAA

how does the addition of the polyA tail occur?

after the cleavage signal of pre-mRNA, polyadenylation occurs of enzymes bound to the CTD of RNA poly II

what happens after splicing and the addition of the polyA tail?

the remaining splicing proteins recruit the Exon Junction Complex (ensures exons successfully joined together), and is then involved in RNA export, localization, and stability.