Eukaryotic Transcription (extended version)

Eukaryotic Transcriptional Initiation

• Two large subunits (similar to beta and beta' in bacterial)
• Two smaller subunits (similar to alpha subunits in bacteria)
• One omega subunit (similar to bacteria)

RNA Polymerase II Dependent Transcription

• The CTD is made up of Tyr-Ser-Pro-Tyr-Ser-Pro-Ser heptapeptide repeats (52 repeats in humans)
• During initiation of transcription, the CTD is unphosphorylated
• As the polymerase begins the elongation phase of transcription, the CTD is phosphorylated
• RNA processing proteins, like capping, elongation, and splicing factors can bind to the CTD depending on it's phosphorylation state

• TFIID consists of a TATA box Binding Protein (TBP), which causes the DNA to bend, and 13 other TBP-Associated Factors (TAFs)

• TFIID forms a complex with TFII A and TFIIB and the TATA-box
• TFIIF and RNAP II then bind to the transcription start site
• TFIIE binds and creates a docking site for TFIIH to bind
• TFIIH has helicase activity which unwinds the DNA
• After transcription has been initiated, TFIIH has kinase activity which phosphorylates the CTD of RNAPII

5' Capping

• This processing occurs co-transcriptionally (i.e when the gene is still actively being transcribed)
• TFIIH phosphorylates Serine 5 on the RNAPII CTD heptapeptide repeat after initiation
• This phosphylation allows the 5' capping enzyme to bind and activate
• Focuses the 5' capping activity to pre-mRNAs transcribed from RNAPII ONLY (not RNAPI or RNAPIII transcripts)

1. Stabilizes the pre-mRNA: protects it from being digested by RNA-digesting enzymes
2. Allows elongation to proceed at a faster rate
3. Serine 2 on the CTD is phophorylated and Negative Elongation Factor (NELF) is released from RNAPII

3' Polyadenylation

1. The 3' end of the mRNA has a AAUAAA sequence. The Cleavage and Polyadenylation Specificity Factor (CPSF) binds to this site.
2. Three more proteins bind to the CPSF-RNA complex: [1] Cleavage Stimulatory Factor (CStF), [2] Cleavage Factor 1 (CF1), and [3] CFII
3. Poly(A) Polymerase (PAP) binds BEFORE the mRNA is cleaved
4. The pre-mRNA is cleaved at the Poly(A) site (downstream of the AAUAAA sequence)
5. The poly(A) Tail is added in two steps
6. PAP adds ~12 A residues slowly
7. PABPN1 binds to this short tail and quickly extends the tail by adding As for a total tail length of ~250 adenines

1. mature mRNA stability
2. export of the mature mRNA to the cytoplasm

Splicing

• hnRNPs function in mRNA processing- Splicing and 3' Polyadenylation

• Group I introns: pre-rRNA, pre-mRNA and pre-tRNA transcripts which use guanosine as a co-factor and use internal base pairing to bring together two exons that must be joined
• Group II introns: pre-mRNA and pre-tRNA which fold into a conserved, complex multi-stem loop structure

• [U-rich small nuclear RNAs (snRNAs)] + [proteins]= snRNPs (small nuclear RiboNuceloprotein Particles)
• snRNPs= U1, U2, U4, U5 and U6
• Each snRNP has a different snRNA, which base pairs with the pre-mRNA transcript

• The ester bond between the intron's 5' phosphorus and the first exon's 3' oxygen is EXCHANGED with an ester bond on the 2' oxygen of the branch point A
• The ester bond between the second exons's 5' phosphorus and the intron's 3' oxygen is EXCHANGED with an ester bond with the first exon's 3' oxygen

Alternative Splicing

• FEMALE Drosophila:
• A promoter that is active only in females transcribes Sxl protein
• The female specific promoter turns off and a male/ female promoter for Sxl is turned on
• The Sxl protein produced from the female specific promoter blocks the spliceosome from accessing the splice site between exon 2 and 3
• Alternative splicing causes exon 3 to be SKIPPED in the final mRNA transcript
• More functional Sxl protein is produced
• MALE Drosophila
• The female specific Sxl promoter is absent. There is no early Sxl protein
• The male/ female promoter for Sxl is turned on
• Without the early Sxl protein, nothing blocks the spliceosome from accessing the splice site between exon 2 and 3
• Exon 3 with the stop codon is INCLUDED in the final mRNA transcript
• No Sxl protein is produced

• FEMALES: the Sxl protein is present and blocks the splice site between exon 1 and exon 2 of the Tra pre-mRNA transcript. Exon 2 is skipped a functional Tra protein is formed.
• MALES: the Sxl protein is absent, so the spliceosome can access the splice site between exon 1 and exon 2. Exon 2, with it's stop codon, is included in the mature mRNA transcript and a functional Tra protein is not formed.

• FEMALES: The Tra protein is present. It binds to Exon 4 on the Dsx pre-mRNA transcript and promotes the splicing of exon 3 to exon 4 and the addition of a poly A tail to Exon 4. This results in a short Dsx protein in females.
• MALES: The Tra protein is absent. Therefore, Exon 4 is skipped. Exon 3 is spliced to Exon 5 and then to Exon 6. This results in a long Dsx protein in males.

mRNA Nuclear Export

• mRNAs bound by hnRNPs= mRNPs
• tRNAs
• ribosomal subunits

• Membrane embedded ring structure with an aqueous center.
• Multiple copies of nucleoporins make up the NPC structure.
• Eight filaments extend into the nucleoplasm to form the nuclear basket. The same occurs on the cytoplasmic side.

• Nuclear Export Factor 1 (NXF1): large subunit
• Nuclear Export Transporter (NXT1): small subunit
• NXF binds to specific proteins associated with mRNPs, for example, RNA export factor (REF) proteins.
• REF proteins are found along the length of the mRNA transcript, 20 nucleotides 5' from every exon-exon junction
• Associates with SR proteins, which are bound to exonic splicing enhancers

• eIF4E: translation initiating factor replaces cap binding protein (CBP)
• PABPC1: replaces PABPN1 on the poly(A) tail
• CBP, PABPN1, NXF1, NXT1 and REF dissociate from the mRNP in they cytoplasm and are recycled back to the nucleus

• Nlp3 is phosphorylated when bound to pre-mRNA
• After the poly(A) tail is added, Nlp3 is dephosphorylated
• Only dephosphorylated Nlp3 can bind to mRNP exporter: QUALITY CONTROL for properly adenylated mRNAs
• Once in the cytoplasm, Nlp3 is phosphorylated again
• Nlp3 dissociates from the mRNP, taking the mRNP exporter with it

Cytoplasmic Processing of mRNAs

1. Primary transcript miRNAs (Pri-miRNAs) are transcribed by RNAPII.
2. Within the pri-miRNA, certain regions fold into hairpin structures that are ~70nt long
3. Drosha, an RNase acts with a double stranded RNA binding protein (DGCR8 in humans/ Pasha in Drosophila) to cut the hairpin out of the pri-miRNA transcript
4. The pre-miRNAs are exported to the cytoplasm by the nuclear export factor exportin 5
5. Dicer, an RNAse processes the pre-miRNA into a mature miRNA
6. One of the miRNA strands is bound by an argonaute protein, forming a RNA-induced silencing complex (RISC)

• Before fertilization: These egg cells have short Poly(A) tails, so only a few molecules of PABPC1 can bind to the tail, thus these mRNAs are not translated efficiently.
• After fertilization: ~150 As are added to the poly A tail, thereby kickstarting translation.

• Before fertilization: CPEB binds to Maskin, which binds to eIF4E. Therefore eIF4E cannot interact with other translation initiating proteins.
• After fertilization: A specific CPEB serine is phosphorylated. Maskin dissociates from the complex and CPSF and PAP cleave the mRNA and extend the Pol(A) tail. More PABPC1 can associate with the longer POly(A) tail, stabilizing eIF4E and its interaction with translation initiating factors.