Lecture Summaries

The semester will begin with general self-introductions by the instructors and student participants. The instructors will then:

1. Present a general overview of the course format, the course website, and assignments,
2. define objectives of the course, and
3. describe what is expected from the student participants.

We will start by giving a biological context for the RNA species that we will be working through in the course—introducing major concepts, techniques, and historical background that will be necessary for the first few sessions. Finally, we will discuss how to critically read scientific papers and access additional resources of the scientific literature.

The role for RNA in protein synthesis of course did not stop with ribosomal RNA. Using radioactively-labeled leucine, Hoagland and colleagues found that although labeled leucine ended up in proteins, it can also become covalently attached to an RNA species. This covalent attachment was dependent upon ATP, and the RNA species interacts with microsomes (ribosomes), providing strong evidence that the RNA was a carrier of amino acids during protein synthesis. Recent reports, like that from Presnyak and colleagues, demonstrate that tRNAs are more than just simple deliverers of amino acids. The availability of tRNAs in cells can influence transcript stability on a global scale.

The papers we have discussed thus far have studied particular systems in great detail. The methods they used can inspire awe in the minds of modern molecular biologists in that simple experiments led to profound insights into fundamental biological principles and made predictions, many of which turned out to be right. Recently, many problems have been approached from the opposite direction: instead of studying one system or one RNA in great detail, study the regulation and activity of many RNA molecules at once to learn about the general principles that govern them. One approach, called high-throughput sequencing, has been very successful and has revealed many interesting properties of gene expression. In this class we will introduce the fundamental principles and methods behind high-throughput sequencing, and particularly focus on high-throughput methods to characterize the abundance and confirmations of RNA species (RNA-seq). Many of the papers we will discuss throughout the remainder of the course will rely on these methods. RNA-seq technology represented an advance towards more precise measurements and comparisons of gene expression, as highlighted in Mortazavi et al., as well as allowed for unbiased ascertainment of mRNA isoform conformations to assess mRNA splicing. In the second paper, we will discuss how RNA-seq can be used as a tool to explore other aspects of RNA biology beyond simple transcript abundance. Wan et al. use a combination of ribonucleases and sequencing to interrogate RNA secondary structure transcriptome-wide.

Although small nuclear RNAs and small nucleolar RNAs (snoRNAs) had been known to exist for years, in the mid 1990s their specific functions remained largely unknown. However, there were preliminary indications. Some, like the snoRNA U3, had been shown to be required for the endonucleolytic cleavages necessary for rRNA maturation. Others had been shown to be involved in methylation of specific bases in rRNA. This week, we will discuss the role of snoRNAs in the deposition of another RNA mark: Pseudouridinylation. In the first paper, Ganot et al. find that pseudouridinylation of specific sites in rRNA molecules is dependent on complementarity between rRNA and snoRNA sequences. In the second paper, Jack et al. show that impairment of this pseudouridinylation system results in ribosomes with reduced activities, an effect that manifests in specific human diseases.

RNA molecules that are antisense to mRNA molecules can act to antagonize the function of their cognate mRNAs. In a classic experiment, Fire and Mello use different combinations of sense / antisense RNAs with complementarity to various regions of specific transcripts and discover that delivery of a double-stranded RNA molecule is a potent repressor of expression. Specifically, injection of either sense or antisense RNA into C. elegans resulted in only modest interference of endogenous genes; however, injection of sense and antisense RNA together resulted in potent interference. These molecules must have complementarity to exonic portions of the gene for maximal effect. This discovery of RNAi (RNA interference) formed the basis for both the use of siRNAs (short interfering RNAs) as experimental tools to specifically (more or less) silence certain genes as well as the possibility of RNA-based treatments for human disease.

Field Trip to Broad Genetic Perturbation Platform (as described in Syllabus).

Although the most glamorous catalytic and structural molecules in the cell are often proteins, any RNA biologist will tell you that RNA molecules have been fulfilling these roles for billions of years. RNA molecules may be ideal scaffolds for regulatory complexes, particularly those that involve the regulation of gene expression, as they may contain both binding sites for many proteins and the ability to hybridize with other nucleic acids. We have seen that RNA molecules have the ability to silence expression at the level of RNA. As described in the first paper, the long noncoding RNA (lncRNA) Xist takes this repression to the level of DNA by repressing gene expression across almost an entire chromosome. By acting as a scaffold for transcriptional repressors and chromatin-modifying enzymes, Xist is able to efficiently shut off transcription of many genes at once. Further characterization of other lncRNAs has led to the possibility that they have many varied functions. As described in the second paper, the Braveheart lncRNA has been observed to activate transcription networks by mediating the epigenetic regulation of cardiac development.

We have seen evidence that RNAs play large regulatory roles in vivo and can be powerful experimental tools. The latest new RNA species to make waves are the CRISPR-associated RNAs (crRNAs), which have recently gained prominence as critical components of the CRISPR-Cas9 genome editing system. The biological basis for the CRISPR-Cas9 system is a naturally occurring prokaryotic adaptive immune process in which the Cas9 protein uses clustered regularly-interspaced short palindromic repeats (CRISPRs) derived from phage or other invading genomes to identify and cleave the corresponding invading DNA sequences. The adaptation of this system within eukaryotic cells and organisms has created a powerful and fast-growing molecular technique that has been used to edit genomes for experimental and potential therapeutic purposes. In the first paper, Cong and colleagues show that Cas9 can be programmed and targeted by crRNAs to direct genome editing in mammals. In the second paper, Hart and colleagues use this technique to target thousands of genes for loss-of-function analysis, finding over 1500 genes that are essential for fitness in human cells.