Eukaryote RNA processing

What are the three main types of RNA processing in eukaryotes ?

5’ capping - addition of a modified guanine cap to the 5’ end

3’-end cleavage and polyadenylation - cleavage of the pre-mRNA followed by addition of a poly(A) tail

RNA splicing - removal of introns and joining of exons

Less prevalent processing reactions include RNA editing, back splicing and RNA methylation

What is the difference between co-transcriptional and post-transcriptional RNA processing ?

Co-transcriptional processing:occurs while RNA polymerase II is still transcribing the gene

Post-transcriptional processing: occurs after transcription has finished

Eukaryotic RNA processing involves both. Splicing can begin before transcription is complete, although some splicing may occur after polyadenylation.

What is a poly(A) tail, and which RNAs normally have one ?

A poly(A) tail is a sequence of adenine nucleotides added to the 3’ end of an mRNA following cleavage of the primary transcript.

Most eukaryotic mRNAs have poly(A) tails

Human mRNA tails are ~250-300 adenines long

Yeast mRNA tails are ~70-80 adenines long

Histone mRNAs are an important exception:they generally do not have poly(A) tails

What is the polyadenylation signal (PAS) ?

The main polyadenylation signal is AAUAAA, although a common variant is AUUAAA

It is located ~21 nucleotides upstream of the cleavage and polyadenylation site

It helps recruit the protein machinery required for cleavage and polyadenylation

What other cis-acting sequences are important for cleavage and polyadenylation ?

Two additional sequence elements are important:

UGUA: found upstream of the AAUAAA signal

G/U-rich region: found downstream of the cleavage site, often containing GUGU repeats

Together with AAUAAA, these sequences help specify where cleavage and polyadenylation occur

What is the difference between cis-acting and trans-acting factors in polyadenylation?

Cis-acting factors: specific RNA sequences within the pre-mRNA, such as AAUAAA, UGUA and the G/U-rich region

Trans-acting factors- proteins that recognise or act on these RNA sequences, such as CPSF, CstF and CFIm

Both are required for efficient cleavage and polyadenylation

What are the two stages of 3’-end processing ?

1) Cleavage: the primary transcript is cut at the polyadenylation site, producing a new 3’ end

2) Polyadenylation: poly(A) polymerase adds adenine nucleotides to the newly created 3’ end

Cleavage is also thought to be necessary for correct termination of transcription

Which proteins recognise the different polyadenylation sequences ?

CPSF - recognises AAUAAA and participates in cleavage

CstF - recognises the downstream G/U-rich region

CFIm and CFIlm - recognises UGUA sequences upstream of AAUAAA

Poly(A) polymerase adds adenine- synthesises the poly(A) tail

These proteins assemble into large RNA-protein complexes (RNPs)

What is the role of CPSF and CstF in cleavage and polyadenylation ?

CPSF (cleavage and polyadenylation specificity factor): recognises the AAUAAA motif and participates in cleavage. It contains four subunits.

CstF (cleavage stimulation factor): recognises G/U-rich sequences downstream of the cleavage site. It contains three subunits, including CstF64 (CSTF2)

How is the poly(A) tail initially synthesised ?

Poly(A) polymerase adds adenine nucleotides to the new 3’ end.

The first ~10 adenines are added individually

The remaining adenines are added rapidly

PABPII helps make the enzyme processive, allowing rapid tail extension

What are the main functions of the poly(A) tail ?

The poly(A) tail has two major functions:

Protection - proteins binding to the tail help protect the mRNA from degradation

Translation efficiency - interactions between poly(A)-binding proteins and translation initiation factors increase the efficiency of translation

What are PABP-I and PABP-II, and where do they act ?

PABP-II: binds the poly(A) tail in the nucleus

PABP-I: binds the poly(A) tail in the cytoplasm

How does the poly(A) tail help increase translation efficiency ?

In the cytoplasm, PABP-I binds the poly(A) tail and interacts with translation initiation factors associated with the 5’cap.

In particular:

PABP-I binds the poly(A) tail

elF4G interacts with PABP-I and the cap-binding proteins binding elF4E

This interaction brings the 5’ and 3’ ends together, making the mRNA appear circularised.

This arrangement promotes efficient translational initiation.

What is the alternative polyadenylation (APA)?

Alternative polyadenylation occurs when a gene has multiple possible polyadenylation sites (PASs)

Using different sites produces mRNA transcripts with different 3’ ends

This can change:

The length of the 3’ UTR

The protein-coding sequence

mRNA stability, translation efficiency and localisation

What are the two main types of alternative polyadenylation ?

1) UTR-APA:alternative polyadenylation within the final exons 3’ UTR

2) CR-APA (coding region APA), also called UR-APA: alternative polyadenylation upstream of the final exon, within the coding region

What is the difference between proximal and distal polyadenylation sites ?

Proximal PAS: located closer to the beginning of the gene

Distal PAS: located further downstream

Using a proximal PAS generally produces a shorter transcript than using a distal PAS

How does UTR-APA affect an mRNA ?

UTR-APA produces mRNAs that encode the same protein but have different 3’ UTR lengths. A shorter 3’ UTR may lose regulatory elements, including:

miRNA binding sites

mRNA localisation signals

Other regulatory sequences

What is the example of UTR-APA involving BDNF ?

Brain-derived neutrophil factor (BDNF) mRNA can have either a short or long 3’ ends UTR

Long UTR (2.85 kb): allows BDNF mRNA to localise to the cell body and dendrites

Short UTR (0.35 kb) results in mRNA being found mainly in the cell body

BDNF is involved in memory formation in the hippocampus. Mice unable to produce the long form show impaired dendritic plasticity.

How does CR-APA affect the protein produced ?

CR-APA can produce mRNAs endowing proteins with different sizes and C-terminal regions.

This happens because alternative cleavage and polyadenylation changes which coding regions are retained in the mature transcript.

Unlike UTR-APA, CR-APA can therefore generate different protein isoforms

How does alternative polyadenylation regulate the production of membrane-bound and secreted IgM?

The IgM heavy chain gene provides an example of CR-APA

Proximal PAS usage: produces the membrane bound form of IgM

Distal PAS usage: produces the secreted form of IgM

The choice of polyadenylation site affects the processing and splicing of the transcript, determining which form is produced

How common is alternative polyadenylation ?

~50-80% of genes in humans, mice and higher plants undergo APA

An average gene showing APA has ~4 putative polyadenylation sites

Most APA sites occur in the 3’ UTR

~5,000 human genes show APA that may alter the encoded protein

What are the main biological consequences of APA ?

Three major potential consequences:

Protein diversity- APA can produce different protein isoforms, as in IgM

Cell proliferation - PAS usage correlates with proliferation and cell division

Disease: cancer cells often use proximal PASs more frequently, producing shorter mRNAs

How does APA differ between proliferating and differentiated cells ?

Proliferating - tend to use proximal PASs, produce shorter 3’ UTRs

Non-proliferating- tend to use distal PASs, produce longer 3’ UTRs

Cancer cells also tend to show increased proximal PASs usage compared with normal cells

What are the three mechanisms that regulate alternative polyadenylation?

Abundance of polyadenylation factors: changing the amount of proteins such as CstF64 can alter which PAS is used

Transcription elongation rate: slower RNA polymerase II elongation can favour proximal PASs usage compared usage

RNA-binding proteins (RBPs); binding to pre-mRNA can alter RNA structure of the accessibility of polyadenylation sites

How does CstF64 concentration regulate APA, using IgM as an example?

In resting or naive B cells:

CstF64 levels are relatively low, formation of the CstF complex is limited, the distal PASs is favoured, producing membrane bound IgM

Following B cell activation and differentiation:

CstF64 concentration increases ~10-20 fold, this allows weaker polyadenylation sites to be recognised, the proximal PASs is used, producing secreted IgM