Mammalian microRNA assembly and function

Published on October 1, 2007 Reviewed on October 13, 2016   26 min

Other Talks in the Series: RNA Interference

My name is Zissimos Mourelatos. I'm an Assistant Professor in the Department of Pathology and Laboratory Medicine at the University of Pennsylvania School of Medicine. Today, I would like to discuss with you some recent aspects of MicroRNP Assembly and Function, and the talk will be divided in three sections. At the first section, we will present an overview of the field. And of the two latter sections, I will present data from our lab on how microRNPs are assembled and function in mammalian systems, and novel assays to profile and detect the localization of microRNAs. The essence of what small RNAs do is presented on this slide.
Small RNAs, which are typically 22 nucleotides in length, assemble with effector complexes known as microRNPs for microRNA ribonucleic proteins, or RISCs for RNA-induced silencing complexes and act as specificity determinants to bring this effector complexes to genes to affect their silencing. Where do these small RNAs come from? There are two sources. One is endogenous genes known as microRNAs, and the other source is small RNAs that are coming from longer double-stranded RNA known as small interfering RNAs or small interfering RNA duplexes. MicroRNAs are initially prescribed as longer transcripts and they are termed pre-microRNAs. These form hairpin structures and by the action of the nuclease Dicer are processed into microRNA duplexes which contain 5-phosphates, and two nucleotide three overhangs. Similarly, Dicer processes small interfering RNAs from longer double-stranded RNA. After this processing step, a single small RNA from the duplexes, either the microRNA duplex or the siRNA duplex shown in red on this cartoon, assembles with Argonaute proteins into effector complexes. So, the Dicer nuclease is the key enzyme that affects processing of small RNAs from their triggered double-stranded RNA which is either pre-microRNA or double-stranded RNA. And Argonaute proteins are the key effector proteins that mediate the function of these small RNAs. Argonaute proteins are large proteins, approximately 95 kilodaltons, and they contain two conserved domains known as Paz and Piwi domains. The Paz domain is approximately 110 nucleotides and binds to the three nucleotide overhang of the microRNA or the siRNA, and the Piwi domain binds to the 5-phosphate and to the first, approximately first seven or eight nucleotides of the microRNA. There are several Argonaute proteins, typically paralogs in each organism. For example, humans contain four canonical Argonaute proteins and four other proteins which are called Kiwi proteins that are similar to the Piwi family of Argonaute proteins of Drosophila. All four canonical Argonaute proteins in humans and in other mammals bind to small interfering RNAs and to microRNAs. Argonaute proteins are conserved and a single Argonaute gene is present in the fission yeast which also contains other components of the RNA interference machinery such as Dicer. Notably, baker's yeast or Saccharomyces cerevisiae do not contain Argonaute proteins, or Dicer, or the RNAi machinery. Argonaute-like proteins exist in Archaea, although their function is not known. Endogenous microRNAs genes are transcribed by