BCH4024 Douma Exam 4 - Lecture 11

What is post-transcriptional modification?

Processing of the primary RNA transcript to create a mature, translation-ready mRNA.

Where does RNA processing occur in eukaryotes?

In the nucleus.

What modifications are made to the 5' end of eukaryotic mRNA?

Addition of a 5' methyl cap.

What modifications are made to the 3' end of eukaryotic mRNA?

Addition of a poly(A) tail.

What is the purpose of the 5' methyl cap?

Protects RNA from degradation, assists in nuclear export, and promotes translation.

What is the purpose of the poly(A) tail?

Protects RNA from degradation and assists with nuclear export and translation.

What are the regions before and at the end of the first and last exons, respectively, of the gene being transcribed that is retained in the final mRNA?

Untranslated regions (UTR)

Why are UTRs not translated?

They do not have protein-coding sequences.

Why are UTRs important if they don't contain protein-encoding sequences?

They contain regulatory sequences that regulate the process of translation (i.e., they determine when translation stops and ends).

What is the C-terminal domain (CTD) of RNA Polymerase II important for?

Recruiting RNA processing enzymes through phosphorylated serines.

What is the 'green flag' for transcription to begin in eukaryotes?

Phosphorylation of the C-terminal domain (CTD).

What enzyme adds the 5' cap on the 5' end of mRNA?

7-methylguanosine

What connects the 5' cap to the mRNA?

A 5'-5' triphosphate linkage.

What is the role of the cap-synthesizing complex?

It binds to the phosphorylated serines on RNA polymerase, waits for the 5' end of the mRNA to be synthesized, and then adds a 7-methylguanosine cap to the 5' end.

What removes the gamma phosphate from the 5' end during capping?

The capping enzyme.

What is the second thing that the capping enzyme does outside of removing the gamma-phosphate?

It uses GTP to add a GMP onto the 5' end of the RNA, releasing a pyrophosphate (PPi) in the process.

Why do all RNA molecules have 3 phosphates at their 5' end?

Because RNA polymerase initiates synthesis de novo, so the first nucleotide's 5' triphosphate is never used to form a phosphodiester bond.

What sequence signals cleavage for polyadenylation?

AAUAAA.

What is the role of polyadenylation factors?

They recognize the transcribed termination sequence, stimulate cleavage of the RNA, and recruit poly(A) polymerase to add the poly(A) tail.

What is the role of the endonuclease with regards to formation of the poly(A) tail?

It cleaves at the poly(A) addition site, 10-30 nucleotides downstream from the sequence 5'-AAUAAA-3' to create a new 3' end for poly(A) polymerase to add the poly(A) tail.

What enzyme adds the A residues to the free 3'-OH group to form the poly(A) tail?

Poly(A) polymerase.

Does poly(A) polymerase require a template? Why?

No. Because it just adds a ton of adenines.

What are exons?

Sequences that code for protein and are retained in the mature mRNA.

What are introns?

Non-coding sequences that are removed from the pre-mRNA.

What defines the 5' splice site of an intron?

A GU sequence.

What defines the 3' splice site of an intron?

An AG sequence.

What is the 'Branch Point A' in an intron?

An adenine residue whose 2'-OH acts as a nucleophile during splicing.

What is the spliceosome?

A large nuclear complex made of proteins and small RNAs that catalyzes RNA splicing.

What are snRNPs?

Small nuclear ribonucleoproteins that make up the spliceosome.

What is the 'business end' of the snRNPs?

RNA. The protein is for structural purposes.

Which snRNP binds the 5' splice site?

U1 snRNP.

Which snRNP binds the branch point A?

U2 snRNP.

Which snRNP contains the catalytic site for splicing?

U6 snRNP.

Which snRNP binds to and sequesters/regulates U6 snRNP?

U4 snRNP.

Which snRNP aligns the pre-RNA for the second splicing reaction?

U5 snRNP.

What happens after the 5' splice site is identified and the spliceosome is ready to splice?

U1 snRNP is placed into the catalytic site of U6, and U4 dissociates from the complex.

What type of reaction happens during splicing?

Transesterification reactions (2 of them)

What happens during the first transesterification reaction in splicing?

The 2'-OH of the branch point A attacks the 5' splice site.

What happens during the second transesterification reaction in splicing?

The 3'-OH of the upstream exon attacks the 5' phosphate of the downstream exon.

What is the lariat structure?

A looped intron released during splicing.

What is alternative splicing?

Different ways of splicing pre-mRNA to create multiple mRNA products from the same gene.

What is an example of alternative splicing?

Exon skipping, alternative 5' splice site, alternative 3' splice site, mutually exclusive exons.

What proteins promote splicing?

SR (serine/arginine rich) proteins.

How do SR proteins promote splicing?

By binding exonic splicing enhancers (ESE).

What proteins repress splicing?

hnRNPs (heterogenous nuclear ribonuclearproteins).

How do hnRNPs inhibit splicing?

By binding to exonic splicing silencers (ESS).

How is the splicing pattern of an mRNA determined?

They are determined by the combination of ESEs and ESSs.

How is mRNA exported from the nucleus?

Cap-binding complex binds to nuclear pore complex and threads mRNA 5' to 3'.

Do prokaryotes process RNA like eukaryotes?

No, prokaryotic RNA is immediately translated without processing.