Non-coding RNA April 11, 2018

Genomce Comparison

Early ideas for the role of RNA What do you the prevailing ideas were regarding the roles of noncoding RNAs?

RNA as Enzymes Tom Cech- Discovered that RNAs can act as enzymes: “RNA can serve as a catalyst and not simply an informational molecule” Read

What do we know about non-coding RNAs as a regulatory molecule in Eukaryotes It has major roles in translation rRNA tRNA Intron splicing 2. It can have catalytic functions 3. It has post-transcription regulatory roles. (microRNAs) 4. It can regulate chromatin structure and function. (long non-coding RNAs)

The discovery of microRNAs 1. When where microRNAs first discovered? Who discovered them? In which model system was microRNAs discovered? 2. What are microRNAs? 3. What are some targets of microRNAs? 4. What type of cellular processes do these regulate?

Caenorhabditis elegans (C. elegans) An elegant worm

Why study worms? Sydney Brenner “Thus we want a multicellular organism which has a short life cycle, can be easily cultivated, and is small enough to be handled in large numbers, like a micro-organism. It should have relatively few cells, so that exhaustive studies of lineage and patterns can be made, and should be amenable to genetic analysis.” --Excerpts from Proposal to the Medical Research Council, 1963

the secret of embryonic development Within this lineage is the secret of embryonic development is the description of the history of each embryonic cell, beginning with the un-cleaved egg. The products of every division are traced until the rudiments of the embryonic organs become distinct. John Sulston

C. elegans has very exciting larval stages  What type of fundamental questions could a person address by studying larva in C. elegans? Larval Stage 1 How do genes regulate growth and developmental changes? How do you find genes involved in growth and development? Larval Stage 1 Larval Stage 1 Larval Stage 1

FORWARD genetic screens to identify genes involved in development and disease

FORWARD genetic screens to identify genes involved in development and disease Randomly mutated genes 1. Find a phenotype! 2. Find the gene that was mutated. This can take a painfully long time!!!

The discovery of the first microRNA (lin-4) What was the question? To find genes involved in growth and development. What type of experiment could they perform? Mutate genes randomly and search for mutants that affected the transitions between the different larval stages. What specific phenotype do you think the researchers were analyzing? Mutants had either blocked transitions into larval stages or mutants that made the transition of larval stages go faster. They did this forward genetic screen and they identified mutants in what they called the lin genes. lin stands for lineage defected.

Results for their screen LOF= loss of function, gene function is disrupted GOF= gain of function, gene is overactive wildtype lin-14 (LOF) lin-4 (LOF) What is lin-14 required for: What is lin-4 required for:

Results for their screen LOF= loss of function, gene function is disrupted GOF= gain of function, gene is overactive wildtype lin-14 (LOF) lin-4 (LOF) What is lin-14 required for: Required for L1 stage What is lin-4 required for: Required to transition from L1- L2 stage

Screen results: Something a little odd LOF= loss of function, gene function is disrupted GOF= gain of function, gene is overactive wildtype lin-14 (LOF) lin-4 (LOF) lin-14 (GOF) What is lin-14 required for: Required for L1 stage What is lin-4 required for: Required to transition from L1- L2 stage What happens when there is too much lin-14? L1 stages repeats

Screen results: Something a little odd LOF= loss of function, gene function is disrupted GOF= gain of function, gene is overactive wildtype lin-14 (LOF) lin-4 (LOF) lin-14 (GOF) Loss of lin-4 looks the same as a gain of lin-14. What does this imply about the function of lin-4?

Screen results: Something a little odd LOF= loss of function, gene function is disrupted GOF= gain of function, gene is overactive wildtype lin-14 (LOF) lin-4 (LOF) lin-14 (GOF) Without lin-4 is the worm making too much lin-14. Is lin-4 inhibiting lin-14?

Results for their screen Graph of LIN-14 and LIN-4 levels in a wildtype embryo L1 L2 L3 L4 Adult LIN-4 LIN-14 Levels Time

Found something interesting Found something interesting. Now they need to find what the lin-4 and lin-14 genes are. Randomly mutated genes Find a phenotype! Find the gene that was mutated. This can take a painfully long time!!!

Results for their screen wildtype lin-14 (LOF) lin-4 (LOF) Found a traditional ORF in the lin-14 genes They found the region of DNA with the mutation, but no start and stop codons.

Results for their screen What did their results suggest about lin-4? wildtype lin-14 (LOF) lin-4 (LOF) Found a traditional ORF in the lin-14 genes They found the region of DNA with the mutation, but no start and stop codons.

Results for their screen Surprisingly, they found that lin-4 did not seem to be made into a protein. Also, they noted that the sequence of the lin-4 gene was complementary to the 3’ UTR of lin-14. A new unexpected cellular regulatory mechanism involving a non-protein-coding transcript had been found! But was lin-4 just some weirdo C. elegans thing. Many thought yes. But… 7 years later another gene identified in a different screen, let-7 was found to function at the RNA level. Victor Ambros

lin-4 does not encode a protein - It encodes for a microRNA Translation blocked!

Let-7 microRNA is highly conserved

What are microRNAs? microRNAs are often called mIR genes derived from precursor microRNAs that are encoded by genes precursor microRNA from hairpin structures