mRNA processing

In prokaryotes, transcription and translation happens …?

simultaneously

Is mRNA processed in prokaryotes?

No.

Where does mRNA processing happen in eukaryotes?

the nucleus

What does it mean for prokaryotes to be polycistronic?

two or more separate proteins are encoded on a single molecule of mRNA. only observed in prokaryotes

Where is the cap on a eukaryotic mRNA?

5’ position

Where is the poly-A tail on a eukaryotic mRNA?

3’ position

What are the 3 general steps of mRNA processing of eukaryotes?

1) 5’ Cap (5’ processing) with 7-methyl guanosine

2) 3’ Poly(A) Tail by polyadenylation (3’ processing)

3) Intron removal (splicing of exons)

What are UTRs?

untranslated regions. located at the 5’ and 4’ ends

What the 5’ UTR function

ribosomal binding site. always upstream of the coding region

What is the 3’ UTR function?

signal for polyadenylation. always downstream of the coding region

What does the phosphorylation of eukaryotic RNA polymerase II allow for?

RNA processing proteins to assemble on it’s tail

What part of the transcript will be part of the protein?

sequences between the 5’ and the 3’ UTR

When is the 5’ cap added?

shortly after transcription when the mRNA is only 20-30 nucleotides long

What is the 5’ cap exactly?

it’s a modified 7-methylated guanosine (at 7’ position it has a methyl group). The methylated guanosine is connected to mRNA via a 5’ to 5’ triphosphate linkage and so the 1st or 2nd nucleotide is sometimes methylated in a pre-mRNA transcript at the 2’ position.

What are the 3 functions of the 5’ cap in eukaryotic mRNA?

1) protects mRNA from 5’ exonucleases (stability)

2) helps in the transport of mRNA to the cytoplasm

3) necessary for ribosome binding in translation (CBP interacts with ribosome)

What is the triphosphate bridge? Why is it important for the 5’ cap?

it’s a super, strong unusual structure that allows for 5’ to 5’ phosphate linkage. Because of its unusual structure, it allows for the 5’ cap to be detected.

How is the Poly(A) tail added to the pre-mRNA through polyadenylation?

1) there is a cleavage of 10-35 NT downstream from the AAUAAA sequence

2) Polymerase II continues to synthesize mRNA into the 3’ UTR region

3) polyA polymerase recognizes the sequence and adds a string of As known as the Poly(A) tail

What are the two functions of 3’ polyadenylation in eukaryotic mRNA?

1) protects mRNA from 2’ exonucleases (stability) similar to telomerase

2) helps in transport of mRNA to the cytoplasm

How does hybridization of mRNA reveal the presence of an intron?

you can use an R-looping experiment, if the double-stranded DNA loops out from the processed RNA then there is an intron

Describe the R-looping experiments?

1) denature the dsDNA gene of interest

2) anneal ssDNA and RNA from the same gene

3) Observe hybrids under EM

Conditions are better for RNA-DNA hybrids. Without introns there is a regular R-loop, with introns there is a ds-looped out region right above it

Why is splicing essential?

prevents deleterious translational defects

What are the 3 essential parts of a mRNA transcript to allow for splicing?

5’ splice site, branch/cryptic A site, 3’ splice site

What are the sequence at each exon that the splicing mechanism looks for?

the “AG” at the end of exon 1 and the “G” at the beginning of exon 2. everything between that is an intron that gets spliced out

What are the steps involved in splicing?

1) the branch site is pulled towards the 5’ end of exon 1.

2) then the 5’ splice site releases its side of the intron and joins the A of the branch site

3) the 3’ splice site releases its side of the intron

4) the lariat shape is formed and gets degraded

What mediates mRNA splicing?

spliceosome complexes

What are spliceosome complexes?

consists of small nuclear RNAs (snRNAs) and proteins (U’s). These form small nuclear ribonucleoprotein particle called snRNPs or “snurps”

What function does snRNPs/”snurps” have?

mediate RNA splicing by base pairing with mRNA sequences that signal splicing (5’ and 3’ splice sites)

What is alternative splicing?

exons from the same gene are spliced in different combinations leading to different mRNAs

Why is the alpha-tropomyosin gene relevant to alternative splicing?

alternative splicing leads to multiple type of mRNAs/proteins from one gene. It produces multiple muscle, and fibroblast mRNA through alternative splicing

When can mRNA exit the nucleus?

when it is completely mature