1. Post-transcriptional regulation of gene expression – 2

2. Figure 4.16 Alternative splicing.

3. Proposed regulation cascade for Drosophila somatic sex determination
Proposed regulation cascade for Drosophila somatic sex determination. Arrows represent activation, while a block at the end of a line indicates suppression. The msl loci, under the control of the Sxl gene, regulate the dosage compensatory transcription of the male X chromosome. (After Baker et al )

4. The differential activation of the sxl gene in females and males
The differential activation of the sxl gene in females and males. (A) In wild-type Drosophila with two X chromosomes and two sets of autosomes (2X:2A), the numerator proteins encoded on the X chromosomes (sis-a, sis-b, etc.) are not all bound by inhibitory denominator proteins derived from genes (such as deadpan) on the autosomes. The numerator proteins activate the early promoter of the Sxl gene. Eventually, in both males and females, constitutive transcription of sxl starts from the late promoter. If Sxl is already available (i.e., from early transcription), the Sxl pre-mRNA is spliced to form the functional female-specific message. (B) In wild-type Drosophila with one X chromosome and two sets of autosomes (1X:2A), the numerator proteins are bound by the denominator proteins and cannot activate the early promoter. When the Sxl gene is transcribed from the late promoter, RNA splicing does not exclude the male-specific exon in the mRNA. The resulting message encodes a truncated and nonfunctional peptide, since the male-specific exon contains a translation termination codon. (After Keyes et al )

5. Drosophila sex determination and alternative splicing
Drosophila sex determination is different from mammals
Depends on chromosome ratio
Presence of Dsx male or female version
An elaborate series of alternative splicing, using splicing inhibitors and activators, dictate which version of Dsx will be produced.

6. Drosophila sex determination and alternative splicing
Rbp1 and Tra2 are SR proteins which may be recruited to Dsx exon 4 splicing enhancers ONLY in the presence of Tra protein
In turn Rbp1/Tra2 recruits U2AF and U2 snRNP to the 3’end of the intron between exons 3 and 4

7. Role of alternative splicing in the perception of sounds of different frequency.
Convergence of Neuronal development, electrophysiology, post-translational modification and alternative SPLICING:
Hair cells are arranged in a particular order
Different hair cells respond to different frequency
Ca+2 activated K+ channel mRNAs are alternatively spliced
Neuronal depolarization activates specific protein kinases
The SR and hnRNP proteins are LIKELY targets of these post-translational modifications
Alternatively spliced mRNAs result

8. Dscam alternative splicing is too complex to explain using any of these examples
However, it seems most of the alternatively spliced forms of Dscam are needed in wild type flies
INTRODUCTION TO POST-TRANSCRIPTIONAL REGULATION
The multiple exons of the Drosophila Dscam gene. The Dscam gene (shown at the top) is 61.2 kb long; once transcribed and spliced, it produces one or more versions of a 7.8-kb, 24-exon mRNA (the figure shows the generic structure of those mRNAs). As shown, there are
several mutually exclusive alternatives for exons 4, 6, 9, and 17. Thus, each mRNA will contain one of 12 possible alternatives for exon 4 (in red), one of 48 for exon 6 ( purple), one of 33 for exon 9 (green), and one of two for exon 17 (yellow). Exons 4, 6, and 9 encode parts of three Ig domains, depicted in the corresponding colors, and exon 17 encodes the transmembrane domain. If all possible combinations of these exons are used, the Dscamgene produces 38,016 different mRNAs and proteins. (Adapted, with permission, from Schmucker D Cell
101:671, Fig. 8.#Elsevier.)
Molecular Biology of the Gene. 7th edition.
Watson, J.D., et al.

9. RNA editing of apo-B pre-mRNA.
Takes place in the nucleus or cytoplasm? Very rare.

10. Balbiani ring mRNPs transporting out of Nuclear Pore Complex
Bruce Alberts, Molecular Biology, 5th Edition

11. Figure Formation of heterogeneous ribonucleoprotein particles (hnRNPs) and export of mRNPs from the nucleus.
(a) Model of a single chromatin transcription loop and assembly of Balbiani ring (BR) mRNP in Chironomous tentans. Nascent RNA transcripts produced from the template DNA rapidly associate with proteins, forming hnRNPs. The gradual increase in size of the hnRNPs reflects the increasing length of RNA transcripts at greater distances from the transcription start site. The model was reconstructed from electron micrographs of serial thin sections of salivary gland cells.
(b) Schematic diagram of the biogenesis of hnRNPs. Following processing of the pre-mRNA, the resulting ribonucleoprotein particle is referred to as an mRNP. (c) Model for the transport of BR mRNPs through the nuclear pore complex (NPC) based on electron microscopic studies. Note that the curved mRNPs appear to uncoil as they pass through nuclear pores. As the mRNA enters the cytoplasm, it rapidly associates with ribosomes, indicating that the 5' end passes through the NPC first.
(a) Model of a single chromatin transcription loop and assembly of Balbiani ring (BR) mRNP in Chironomous ten tans. Nascent RNA transcripts produced from the template DNA rapidly associate with proteins, forming hnRNPs. The gradual increase in size of the hnRNPs reflects the increasing length of RNA transcripts at greater distances from the transcription start site. The model was reconstructed from electron micrographs of serial thin sections of salivary gland cells. (b) Schematic diagram of the biogenesis of hnRNPs. Following processing of the pre-mRNA, the resulting ribonucleoprotein particle is referred to as an mRNP. (c) Model for the transport of BR mRNPs through the nuclear pore complex (NPC) based on electron microscopic studies. Note that the curved mRNPs appear to uncoil as they pass through nuclear pores. As the mRNA enters the cytoplasm, it rapidly associates with ribosomes, indicating that the 5' end passes through the NPC first.

12. Model of transporter passage through an NPC.
mRNP exporters. Interact with FG domain of FG nucleoporins on one hand and REF on the other hand.
SR proteins. Part of EJC.

13. Remodeling of mRNPs during nuclear export.
CBC Vs eIF4E
PABPII Vs PABPI

14. Reversible phosphorylation and direction of mRNP nuclear export.
Coupling of polyA processing and export:
Np13 is a SR protein
Phosphorylated on pre-mRNA
Dephosphorylated after polyA processing
Exporters can ONLY bind to dephosphorylated Np13

15. Export of mature mRNAs is linked to spliceosomal complex assembly
Mutate one conserved residue at either the 5’-3’junction or 3’- 5’junction – no export
Mutate both - export
Thalassemia

16. Figure 8.24 Transport of HIV mRNAs from the nucleus to the cytoplasm.
RRE bound Rev proteins interact NXF1/NXT1 with very high affinity allowing transport of UNSPLICED RNAs

18. Figure 8.32 Discovery of nonsense-mediated mRNA decay (NMD)

19. miRNA and siRNA

21. Introduction of extra copies of the gene did not produce gain of function rather led to loss of function
They could not explain the phenotype
A similar phenomenon was observed in Neurospora Crassa in 1992 by Romano and Maciano
First report of RNA interference in animals came from Kemphues laboratory in Either sense or anti-sense strand would degrade the message for par-1 in C. elegans

23. lin-4 transcript is only 22 or 61 nucleotide long
It blocks expression of LIN-14 protein
lin-14 3’UTR has 7 repeat sequences that are complementary to lin-4 sequence
They did not address the mechanism of action in this paper

24. The Small RNA Revolution

25. The paper that earned one of the fastest the Nobel Prizes in the history

26. Andrew Fire’s Nobel lecture

27. Andrew Fire’s Nobel lecture

28. Andrew Fire’s Nobel lecture

29. Andrew Fire’s Nobel lecture

30. Is the interfering RNA a "contaminant" with stable structure?
Two puzzles to investigate:
• How could both "Sense" and "Antisense" RNA produce interference?
• Why should injected RNAs outlast normal mRNAs in the same embryo?
Is the interfering RNA a "contaminant" with stable structure?

31. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans - Andrew Fire, SiQun Xu, Mary K. Montgomery, Steven A. Kostas, Samuel E. Driver & Craig C. Mello - NATURE | VOL 391 | 19 FEBRUARY 1998, pp 806

32. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans - Andrew Fire, SiQun Xu, Mary K. Montgomery, Steven A. Kostas, Samuel E. Driver & Craig C. Mello - NATURE | VOL 391 | 19 FEBRUARY 1998, pp 806

33. RNA interference affects mRNA abundance
No dsRNA, Mex3 probe
No dsRNA, no probe
as RNA, Mex3 probe
dsRNA, Mex3 probe
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans - Andrew Fire, SiQun Xu, Mary K. Montgomery, Steven A. Kostas, Samuel E. Driver & Craig C. Mello - NATURE | VOL 391 | 19 FEBRUARY 1998, pp 806

34. Andrew Fire’s Nobel lecture

35. They reported actual decrease in RNA levels (mex3 experiment)
The authors inferred that it must be a post-transcriptional mechanism as dsRNA corresponding to introns did not produce any phenotype
They reported actual decrease in RNA levels (mex3 experiment)
dsRNA effect goes beyond a cell and can be transmitted to the next generation
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans - Andrew Fire, SiQun Xu, Mary K. Montgomery, Steven A. Kostas, Samuel E. Driver & Craig C. Mello - NATURE | VOL 391 | 19 FEBRUARY 1998, pp 806

36. Figure 8.25 Base pairing with target RNAs distinguishes miRNA and siRNA.

37. Figure 8.26 miRNA processing.

38. A common pathway for
generating siRNAs and
miRNAs (both require
Dicer and RISC)

39. Experimental Figure 8.27 miRNA function in limb development.