BCH Graduate Survey of Biochemistry

Transcription

1 BCH 5045 Graduate Survey of Biochemistry Instructor: Charles Guy Producer: Ron Thomas Director: Marsha Durosier Lecture 29 Slide sets available at:

2 David L. Nelson and Michael M. Cox LEHNINGER PRINCIPLES OF BIOCHEMISTRY Fifth Edition CHAPTER 26 RNA Metabolism 2008 W. H. Freeman and Company

3 Alternative processing of rat calcitonin gene transcript where the primary transcript has two poly(a) sites; one that predominates in the brain, and the other in the thyroid. So, function with respect to processing is not solely limited to the coding region transcripts.

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5 Processing of pre-rrna transcripts in bacteria.

6 Processing of pre-rrna transcripts in vertebrates.

7 snornas guide methylation reactions of rrna modification through RNA pairing.

8 trnas processing in bacteria and eukaryotes.

9 MicroRNAs (mirnas) are highly conserved small RNAs encoded in the genomes of plants and animals. The mature ~21-mer RNAs regulate gene expression by binding to the 3'-untranslated regions (3'-UTR) of specific mrnas. The primary mirnas transcript is a larger RNA of variable length, a pri-mirna. Processing is mediated by two endoribonucleases in the RNase III family, Drosha and Dicer. In the nucleus, the pri-mirna is reduced to a 70 to 80 nucleotide precursor mirna (premirna) by a protein complex containing Drosha. After the pre-mirna is exported to the cytoplasm, it is acted on by Dicer to produce the nearly mature mirna paired with a short RNA complement. The complement is removed by an RNA helicase, and the mature mirna is incorporated into an RNA-induced silencing complex (RISC), which then bind a target mrna, and promote their destruction.

10 Hammerhead ribozyme that promote site-specific RNA cleavage reactions associated with replication. Their name comes from the view of secondary structures that look like they are shaped like the head of a hammer.

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13 The Central Dogma is turned upside down. How to account for RNA viruses? Is it possible in nature to go from RNA to DNA? Howard Temin and David Baltimore in 1970 would independently discover reverse transcriptase that can catalyze the synthesis of DNA from an RNA template.

14 Vidarabine Acyclovir Drug Gancyclovir and Valcyte (valganciclovir) Nucleoside-analog reverse transcriptase inhibitors (NRTI): AZT (Zidovudine), ddi (Didanosine), ddc (Zalcitabine), d4t (Stavudine), 3TC (Lamivudine) Non-nucleoside reverse transcriptase inhibitors (NNRTI): Nevirapine, Delavirdine Virus Type Herpesviruses Herpes simplex (HSV) Cytomegalovirus (CMV) Retroviruses (HIV) Retroviruses (HIV) Many viruses have evolved specialized polymerases to preferentially replicate viral nucleic acids at the expense of the host nucleic acids. Fortuitously, the unique properties of many viral polymerases provide a potential specific target for antiviral agents. This has yielded a means to find a number of Chemical Type Nucleoside analogue Nucleoside analogue Nucleoside analogue Nucleoside analogue Nucleoside analogue Target Virus polymerase Virus polymerase Virus polymerase Reverse transcriptase Reverse transcriptase specific antiviral drugs currently in use. Many of these antiviral drugs function as polymerase substrate nucleoside/nucleotide analogues.

15 In bacteria the chromosome is circular, but in eukaryotes the DNA in a chromosome is linear. In the replication of the bacterial chromosome, when it comes time to remove the RNA primers and fill in the gaps, there is always a piece of DNA 5' to the gap that must be filled in. In eukaryotes this is true until replication reaches the ends of the chromosome. If a RNA primer resides at the very 5'-end, then excision and fill in cannot occur. Each time the chromosome is replicated, it will be missing the end and grow progressively shorter. The solution to this problem is the enzyme known as telomerase. At the end of the chromosome is the telomere, which has a special structure and sequence. The sequence consists of many short repeats (1000 or more TTAGGG hexamer repeats).

16 Telomerase contains an RNA component that binds to the 3' end of the DNA, then the enzyme acts as a special type of reverse transcriptase and synthesizes a DNA strand using the RNA as a template. By the action of telomerase, a chromosome could grow indefinitely, but it generally does not. In somatic cells, the chromosomes usually show a shortening with time presumably because telomerase is not expressed and is not active in these cells. Differential regulation of telomerase activity in normal and oncogenic cells is a strategy for the design of new classes of telomerase inhibitors. The telomerase enzyme antagonist GRN163L is a lipidmodified 13-mer oligonucleotide N3V P5V-thiophosphoramidate, that is complementary to the template region of telomerase RNA (htr). The Geron company plans four Phase 2 clinical trials of GRN163L also known as imetelstat in 2010 on non-small cell lung cancer and breast cancer, multiple myeloma and essential thrombocythemia.

17 This slide is not in the lecture video At the Jefferies 2011 Global Healthcare Conference in June in New York City, an update on the Imetelstat Phase 2 clinical program was provided. Patients have been enrolled in two large randomized clinical trials of patients with non-small cell lung cancer and breast cancer. Progress on the trials is ahead of projections.

18 Figure 1. Telomeres are repetitive DNA sequences at the end of linear chromosomes. In most normal cells, progressive telomere shortening is observed each time a cell divides. When telomeres are short, cells stop dividing and undergo a growth arrest (called replicative senescence). Almost all cancer cells are immortal, having overcome cellular senescence by reactivating or upregulating telomerase, a cellular reverse transcriptase that stabilizes telomeres. In this figure, human dermal BJ fibroblasts at low passage, population doubling (PD) 16 and 61, were treated with colcemid to arrest cells in mitosis and chromosome spreads were made. Samples were prepared for quantitative fluorescence in situ hybridization (Q- FISH) microscopy using Cy3-labeled peptide nucleic acid probes specific for (TTAGGG)n telomere sequences (red/pink) and the general DNA dye DAPI (blue/purple). Images of Cy3 and DAPI fluorescence were acquired on a digital image microscopy system to calculate the fluorescence intensity for each telomere. The telomere length is proportional to the number of hybridized probes. This figure is from Shay et al., (2001) Telomerase and cancer. Hum Mol Genet 10:

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22 Gene Silencing by RNAi Initially what has become known as PTGS or interference RNA (RNAi) was a unusual genetic phenomenon observed in petunias and a few other plants. In the early 1990s, Carolyn Napoli, Rich Jorgensen and colleagues introduced a pigment-producing gene under the control of a strong promoter hoping to get more intense flower color. However, many of the flowers appeared variegated or even white. The phenomenon was termed "cosuppression", since the expression of both the introduced gene and the homologous endogenous gene were suppressed. Similarly, introduction of a viral gene for the TEV virus coat protein resulted in specific resistance to viral infection in transgenic plants. Lindbo et al., 1993 proposed that the resistant state and reduced steady state levels of transgene transcript accumulation were mediated at the cellular level by a cytoplasmic activity that targets specific RNA sequences for inactivation.