MBB222 - Lecture 29/30: Eukaryotic transcription and processing (pt.2)

what does hnRNA (pre-mRNA) acquire when processed to mRNA

• 5’ cap

• 3’ poly-A tail

• spliced as it is processed into mRNA

why is the 5’ cap added early on before the synthesis of the primary mRNA is done

to protect the pre-mRNA from degrading - helps in early processing steps (i.e. splicing) and ensure proper translation later

why is the 5' cap only added to RNA polymerase II (mRNA) transcripts

RNA polymerase II has a unique tail structure that physically carries and recruits the capping enzymes

how does the 5’ cap help in initiating protein synthesis

5’ cap helps in the binding of mRNA to ribosomes → initiates protein synthesis

how can mRNA capping also stabilize the mRNA

by protecting it from the nuclease

what are the mRNA processing enzymes associated with

C-terminal domain (CTD) of RNA polymerase II

which enzymes allows processing to occur as the transcript is being synthesized

• capping factors / enzymes

• enzymes / factors involved in splicing

• polyadenylation enzymes

what happens during transcription termination and poly A tailing (polyadenylation)

• enzyme complex (includes: endonuclease, polyadenylate polymerase, other proteins) binds to both the cleavage signal sequence in the mRNA + C-terminal domain of the RNA polymerase II

• endonuclease cleaves the transcript 10-30 bases downstream from the 3’ end of the sequence - AAUAA

• polyadenylate polymerase adds 80-250 A residues (poly A tail)

why is the 3’ polyA tail important for mRNA

• helps in the export from the nucleus → ensure only fully processed mature messages reach the cytoplasm

• protein translation → forms a loop that helps ribosomes efficiently restart and read the message over again

• stability → long ends of adenosines acts as a buffer against cell enzymes (nucleases) that degrade RNA

how can you purify mRNA away from tRNA and rRNA

by exploiting the unique poly(A) tail found on most mRNA → mRNA will hybridize to beads with covalently attached poly T RNA segments (A-T binding)

why is it difficult to purify any one type of mRNA out of a mixture of many RNA’s

• chemical similarity → identical backbones / shared features (i.e. polyA tails)

• high instability / degradation

• size and conformation → mRNA molecules can fold into complex + tight secondary / tertiary structures - overlapping or aggregation can occur to prevent clean separation

where is eukaryotic mRNA processed

in the nucleus

what happens in the nucleus

• intro sequences are removed by splicing

• 5’ ends are protected with a m7G cap

• polyAtail added at 3’ end → facilitates mRNA translation

what are introns

non-coding regions

what are exons

coding regions

which (exon or intron) are initially transcribed into pre-mRNA (hnRNA)

both are initially transcribed

which (exon or intron) are excised

introns are excised - exons are covalently linked to form the mature mRNA → process called splicing

why does splicing reduces the size of mRNA substantially

removes large non-coding sections (introns) while keeping only the protein-coding sections (exons)

how was splicing first detected by R-looping

• ssDNA was hybridized to its corresponding mRNA

• large loops of DNA were observed - did not anneal

• loops encode introns that are excised out of the pre-mRNA to make the mRNA molecule

what is the mechanism of spliceosome-mediated splicing of precursor mRNA

• spliceosome performs the cleaving

• cleavage of 5’ splice site and formation of lariat ( cowboy - lasso ) RNA structure

• cleavage of 3’ splice site and ligation of splice exons

what is the branch point in splicing

a specific nucleotide sequence inside an intron that contains a key adenine residue - acts as the chemical attack point to help remove non-coding RNA

what is the highly conserved nucleotide in the precursor mRNA transcript at the:

• 5’ end of the intron

• branch point

• 3’ end of the intron

GU / A / AG

draw out the reaction of the spliceosome-mediated splicing of precursor mRNA

identify:

• identify the two transesterification reactions

• identify exon-intro junctions (5’ + 3’ splice sites)

• identify the branch point A

• be able to draw a lariat intermediate

describe how the spliceosome assembly forms

• U1 snRNP → recognizes and binds to the conserved 5’ splice site on the pre-mRNA

• U2 snRNP → attaches to the branch point sequence containing a conserved adenosine near the 3’ end of the intron

• U4 / U5 / U6 → complex joins the assembly - loops the intron and brings the splice sites into proximity

• U1 / U4 → dissociates and allows U6 to pair with U2 and the 5’ splice site → creates an active RNA - based catalytic center with metal ions

describe the catalytic steps (transesterification)

• 2 OH groups of the branch point adenosine attacks the 5’ splice site phosphate - cuts the 5’ exon free and forms a looped intermediate (intron lariat)

• newly freed 3’OH group of the 5’ exon attacks the 3’ splice site at the boundary of the next exon

• two exons join together into mature mRNA - excised intron lariat is released and degraded

why is the U2 base pairing with branch site mRNA sequence important in spliceosome mediated splicing

forces the specific branchpoint adenosine to bulge outward from the RNA helix → exposes its 2 OH groups to attack the 5’ splice site

what does self-splicing require

• pre-mRNA

• magnesium (mg 2+)

• free G [ Guanosine cofactor ] → GDP, GMP, GTP, guanosine

which group of introns are spliceosome independent (self-splicing)

group I / group II introns

describe how group I self-splices

• 3’ OH of an exogenous guanosine cofactor (G) participates in a transesterification reaction with the phosphoryl group at the 5’ end of the intron

what is the difference between group 1 and group 2 intron self-splicing

group 1 introns:

• uses an exogenous guanosine cofactor

• excised intron is linear

group 2 introns:

• uses an internal adenine nucleotide

• forms a lariat shape

why are introns and alternative splicing important

• splicing at alternative splice sites to include/exclude different exons can lead to the production of several protein products from one gene

• is a powerful mechanisms for expanding the diversity of genomic sequences - one gene can produce multiple products

• alternative splicing can be regulated → gives differential splicing at different times

what are isoforms

alternative splice products from the same pre-mRNA

how are isoforms formed by alternative splicing

all introns are removed while some exons can also be removed along with the introns → allowing the formation of distinct mRNA molecules that code for proteins with different structures / jobs