Post-transcriptional regulation of gene expression – 2 17-10-2017

Figure 4.16 Alternative splicing.

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. 1987.) http://www.ncbi.nlm.nih.gov/books/NBK10025/

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. 1992.) http://www.ncbi.nlm.nih.gov/books/NBK10025/

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.

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

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

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. 2000.Cell 101:671, Fig. 8.#Elsevier.) Molecular Biology of the Gene. 7th edition. Watson, J.D., et al.

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

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

Figure 8.23 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.

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.

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

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

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

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

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

miRNA and siRNA

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 1995. Either sense or anti-sense strand would degrade the message for par-1 in C. elegans

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

The Small RNA Revolution

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

Andrew Fire’s Nobel lecture

Andrew Fire’s Nobel lecture

Andrew Fire’s Nobel lecture

Andrew Fire’s Nobel lecture

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?

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

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

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

Andrew Fire’s Nobel lecture

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

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

Figure 8.26 miRNA processing.

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

Experimental Figure 8.27 miRNA function in limb development.