RNA-protein interaction Students: Guo Xiaoyong Fan Wenzhu Liu Yunhui

一. Background DNARNA proteins transcriptiontranslation proteins RNA and proteins are two kinds of most important molecules in cells. The interaction between proteins and RNA is one of the key issues in molecular biology.

RNA, in its varied forms,interacts with proteins to carry out fundamental roles in the cell.

(2) mRNA 、 rRNA & tRNA in translation Translation proceed in cytoplasm in an ordered process.It includes three phages.It requires free am- ino acids,free energy,mRNA,tRNA, Ribosomes and nonribosomal fac- tors(eIF in Eukaryotes and IF in some prokaryotes).It should be no- ted that the polypepetide sequence is in total agreement with gene co- de since tRNA anticodons are com- plementary of mRNA codons and the mRNA sequence is a mirror of the gene sequence.

(4) sRNA and protein Small regulatory RNAs, including siRNA, miRNA,piRNA,and hsRNA.commonly referred to as RNA silencing, such as RNA interference (RNAi), translational repressi -on, and heterochromatin formation in all higher eukaryotes and play important roles in cellular processes as diverse as develop- ment,stress response, or transposon silenc- ing. Soon after the discovery of small

Understanding the contributions of various RNA to the control of translation (protein synthesis on the ribosome) in the cell forms an important theme within the structural biology and biophysics group. regulatory RNAs, members of the RNase III familyand Argonaute protein family, some RNA binding proteins, and etc, were identified as their major cellular protein interactors and involved in the biogenesis and various cellular functions of small RNAs.

二. Outlook and further perspectives RNA-protein interactions are a central component of posttranscriptional regulation at multiple levels including RNA processing, transport and translation. The sequenced Human genome reveals hundreds of potential RNA binding proteins. A critical step towards understanding the function of RNA binding proteins is to identify and determine how they interact with their target RNAs.

三. The article

1.Introduction Telomerase is an essential cellular ribonucleoprotein that solves the end replication problem and maintains chromosome stability by adding telomeric DNA to the termini of linear chromosomes. Here we use a single molecule approach to dissect the individual assembly steps of telomerase.

Direct observation of complex formation in real time revealed two sequential steps of protein-induced RNA folding, establishing a hierarchical RNP assembly mechanism: interaction with the telomerase holoenzyme protein p65 induces structural rearrangement of telomerase RNA, which in turn directs the binding of the telomerase reverse criptase to form the functional ternary complex.

(1) Fluorescence resonance energy transfer (FRET) FRET is a process in which an excited fluorophore (the donor) transfers its excited state energy to a light absorbing molecule (the acceptor). This transfer of energy is non-radiative, due to a dipole-dipole interaction between the donor and acceptor. 2.Methods

(2) Construction of FRET-labeled telomerase RNA by DNA-splinted RNA ligation

Cy5 Cy3 >10nm Cy5 Cy3 <10nm

3.Results & Analysis Full-length telomeraseRNA labelled with FRET donor (Cy3) and acceptor (Cy5). RNA molecules wereimmobilized on a streptavidin-coated surface by a biotin molecule engineered onto an extension of stem II. The interaction sites with p65 (red) and TERT (green) are highlighted.

FRET histograms of RNA molecules in the absence ofprotein (grey bars),the presence of 10nM p65 (red bars) or 10nM p65 plus32nM TERT1–516 (green bars).

By real-time observating,the author found that addition of purified p65 give rise to a second population.p65 did not alter fluorescence intensities from the RNA singly labelled with Cy3 or Cy5 at the same locations.

To investigate the assembly of p65–RNA– TERT ternary complex,the authours used a purified TERT polypeptide containing the amino-terminal 516 amino acids. A single-molecule FRET trajectory showing hierarchical RNP assembly after the addition (black arrow) of a protein mixture of p65 (10 nM) and TERT1–516 (10 nM), characterized by a p65-induced FRET change (red arrow) followed by a second FRET transition after complete assembly of the ternary p65–RNA–TERT1–516 complex (green arrow).

These results indicate RNP’S assemblye is initiated by p65 binding, stabilizing a RNA structural intermediate, which in turn promotes thefunctional co-assembly of TERT with telomerase RNA.

To characterize the p65-induced assembly intermediate structurally,the authors generated a series of truncated constructs composed of telomerase RNA stems I and IV. GA

From the above charter,the authors deduce that the p65-induced RNA conformational change occurs within stem IV and requires the central stem IV GA bulge, which is conserved across all Tetrahymena species.

4.Conclusion These experiments show a hierarchical assembly mechanism for telomerase RNP in which the protein subunits mould a specific RNA tertiary structure in a stepwise fashion.The protein p65 induces a structural change within the stem IV region of the RNA.The RNA conformation in the p65–RNA complex is further altered by binding of TERT, resulting in a compact RNA tertiary fold within the functional telomerase RNP.