CH29: rna function, biosynthesis, & processing (2)

29.4-29.5

True or False: virtually all transcription products are further processed in eukaryotes, what about in prokaryotes?

True for euk, but some prok transcripts are modified

what is pre-mRNA and what 2 things happne to pre-mRNA molecules?

• pre-mRNA: immediate product of RNA polymerase II

• pre-mRNA molecules:

  • are spliced to remove introns

  • undergo modifications to both 5’ and 3’ ends

what is the 5’ cap? what 2 things do they do?

• 5’ cap: distinct terminus at 5’ containing an unusual 5’ - 5’ triphosphate linkage

  • protects 5’ ends from phosphatases & nucleases, contributing to mRNA stability

  • enhances mRNA translation by eukaryotic protein-synthesizing systems

what is the poly(A) tail and how is it added? what can it do?

• poly(A) tail: a string of ~250 adenine nucleotides at the 3’ end

  • added by poly(A) polymerase after cleavage of pre-mRNA by endonuclease

• may enhance translation efficiency & mRNA stability

what are the 3 steps to form cap 0?

1. trimming: one phosphate is clipped off the front of new RNA (hydrolysis)

2. attaching: the RNA 5’ end atks α-phosphorous atom of GTP to form 5’ cap

3. decorating (methylation): methyl group is added to nitrogen at the 7th position of new guanine (N-7) to form cap 0

   1. adjacent riboses can be methylated to form cap 1 or cap 2 to help cell distinguish its own RNA from viral RNA

what does it mean when primary transcripts in eukaryotes are polyadenylated?

• it means a long tail of adenine nucleotides (poly A tail) is added to the 3’ end of RNA mc after it has been transcribed from DNA

what is RNA splicing?

process by which introns are excised and exons are linked to form the final mRNA

what are the common structural motifs of introns that mark the beginning and end?

• begin with GU (5’ splice site) and end with AG (3’ splice site)

describe the consensus sequence found at the 3’ end of a vertebrate intron

• 3’ end has polypyrimidine tract (~10 pyrimidines), followed by sequence NCAG, where AG marks actual end of intron

where is the “branch site” located and why is it significant?

• branch site: 20-50 nucleotides upstream of 3’ splice site

• important bc it is the point where the 5’ end of the intron attaches during the splicing process to form a loop structure

describe the 2 sequential steps of the splicing reaction (transesterification), what intermediate does it generate?

1. lasso: branch site adenine atks 5’ splice site, freeing exon 1 and forming lariat (2)

2. stitch: newly freed 3’-OH of exon 1 atks 3’ splice site, joining 2 exons and releasing intron

generates a branch and forms a lariat (loop) intermediate

Consider the sequence 5–AGGUAAGU(N)₂₄₅(Py)₁₀NCAGG–3′.

How long is the intron in this sequence?

start at GU, GUAAGU = 6, N₂₄₅= 245 nucleotides, Py₁₀= 10 pyrimidines, NCAG end at AG = 4

6+245+10+4 = 265

what are snRNPs, and which specific ones are considered essential for the splicing of mRNA precursors? what about them is highly conserved?

• small nuclear ribonucleoprotein particles: complexes formed by small nuclear RNAs (snRNAs) and specific proteins

  • essential ones: U1, U2, U4, U5, U6

  • snRNAs’ secondary structures are highly conserved bc their specific folded shapes are critical for recognizing splice sites

how is the 60S spliceosome formed, and what is its role?

• formed by association of essential snRNPs, hundreds of splicing factors, & pre-mRNA

• role: catalyze precise excision of introns and ligation of exons

describe the roles of snRNPs for U1, U2, U5, U4, U6

snRNP Size of snRNA (nucleotides) Role

U1 165 Binds the 5' splice site

U2 185 Binds the branch site

U5 116 Binds the 5' splice site and then the 3' splice site

U4 145 Masks the catalytic activity of U6

U6 106 Catalyzes splicing

what are the 6 steps of the splicing process?

• The Assembly

1. U1 binds to the pre-mRNA 5’ splice site

2. U2 binds intron branch site (req energy from ATP)

3. U4-U5-U6 tri-snRNP binds complex of U1, U2, and mRNA precursor to form spliceosome

• The Activation

4. spliceosome rearrange, kicking out U1 & U4, allowing U6 to twist (intramc rearr) and pair with U2 to make catalytic center that will perform cut (interact w/ 5’ end of intron)

• The Execution

5. First cut: 5’ exon is snipped off, intron forms lariat loop

6. Final stitch: U5 lines up 2 exons (5’ w 3’) and they will go transesterification to glue tgt

   1. mature mRNA released, leaving spent snRNP, U2, U5, U6 still attached to lariat intron

what is the difference between a “cis-acting” and a “trans-acting”’ mutation in the context of splicing disease?

• cis-acting mutation happens within the pre-mRNA sequence (eg. at splice site), affecting only that specific transcript

• trans-acting mutation occurs in splicing machinery (eg. snRNP protein), which could affect splicing of many diff transcripts

what are some thalassemias (diseases from defective hemoglobin synthesis) caused by? what about retinitis pigmentosa?

• thalassemias: caused by mutations at the 5’ or 3’ splice sites in either of the two introns of hemoglobin beta chain or in its exons

• retinitis pigmentosa: disease of acquired blindness is due to mutation in pre-mRNA splicing factor that is part of U4-U5-U6 tri-snRNP

what is alternative splicing and what could it lead to?

• alternative splicing: mechanism by which diff combo of exons from same gene may be spliced into a mature RNA

  • generates protein diversity and leads to changes in coding sequence

what is splice site selection determined by?

• determined by the binding of trans-acting splicing factors to pre-mRNA cis-acting sequences

what are some human disorders attribuuted to defects in alternative splicing?

what are transcription and mRNA processing coordinated by?

• the CTD (carboxyl terminal domain) of RNA polymerase II

  • CTD has repeated seq YSPTSPS, where S2, S5 or both may be phosphorylated

  • CTD caps enzymes, are components of splicing machinery and are endonuclease that cleaves transcript at poly(A) addition site

describe the structural transition a microRNA undergoes from its precursor form to its active regulatory form

• it starts as a large transcript that folds into a hairpin structure, which is then cleaved by specific nucleases into short, single-stranded form that binds to regulatory proteins

what are microRNAs?

• small (20-23 nucleotides) RNAs that regulate gene expression in eukaryotes

what are ribozymes?

• RNAs that function as catalysts

what are group I self-splicing introns? what do they require as a cofactor?

• introns that can excise themselves

• initially identified in rRNA from tetrahymena

• require guanosine as a cofactor

describe the transesterification reaction mechanism of group I self-splicing intron, how does its released form differ from spliceosome-mediated splicing?

• it aligns splice sites using an internal guide sequence (IGS) that base-pairs with exons (base pair between IGS in intron and 5’ & 3’ exons)

• then a phosphodiester bond is formed between 2 exons and intron is release

• unlike spliceosome-mediated splicing which releases a lariat, group I introns are released as linear molecules

in self-splicing mechanisms, how is the catalytic site formed?

• by the intron itself

in group II self-splicing introns, what is the attacking group?

• atking group is a 2’-OH of an adenylate in the intron

• intron released is released in lariat form

in vitro experiments, there’s the possibility of self-ligating ribozymes, what does this mean?

• RNA molecules capable of joining other short RNAs to their own ends

How does group II self-splicing resemble spliceosome-catalyzed splicing of mRNA precursors? (choose all that apply )

a. A ribose hydroxyl group attacks the 3′ splice site and the

newly formed 5′-OH terminus of the upstream exon then

attacks the 5′ splice site, form a phosphodiester bond.

b. Both reactions are transesterifications with the phosphate

moieties at each splice site retained in the products.

c. The attack at the 5′ splice site is carried out by a part of the

intron itself.

d. The number of phosphodiester bonds increases.

e. The intron is released in the form of a lariat.

b, c, e