Lecture 20: Regulation of Translation II

Regulation of Translation II Regulation of translation initiation at the 3’ UTR in eukaryotes • 3’ UTR can be quite large and their regulation is common, particularly during development ◦Differences in translation levels due to different 3’-UTRs sequence elements and can be visualized with a GGP reporter in C. elegans • Elements within 3’ UTR recruit specific cellular factors ◦eIF4E-binding proteins (4E-BPs) that compete with eIF4E, which disrupt formation of closed loop mRNA initiation complex ◦Factors that interact with mRNA decay machinery Polyadenylation levels can be regulated by 3’ UTR binding proteins • Translation depends on 3’ UTR length, which is regulated by 3’ UTR binding proteins ◦In Xenopus laevis oocytes, maternally-derived mRNAs are not initially translated - they are dormant, or translationally repressed ◦Dormant mRNAs have short poly(A) tails, which become extended during maturation, allowing their translation ◦The 3’ UTR of dormant mRNA has sequences called cytoplasmic polyadenylation elements (CPEs) and when they are binds by a protein called CPEB, translation is repressed ‣ CPEB interacts with Maskin and Maskin interacts with eIF4E. This stops formation of the closed loop required for translation initiation, thereby “masking” the expression of mRNA ‣ Maskin binds to eIF4E through a conserved sequence element (YXXXXLphi) which mimics an element in eIF4G. Maskin is a type of 4E-BP • CPEB is phosphorylated by Eg2 kinase, which allows it to recruit CPSF to an AAUAAA polyadenylation signal • CPSF recruits poly(A) polymerase (PAP) and this extends the poly(A) tail • Poly(A) binding protein (PABP) can then bind to the poly(A) tail, allowing eIF4G to bind and displace maskin, now forming the initiation complex allowing translation to proceed Translation can be further regulated following polyadenylation • 3’ UTR sequence elements recruit a variety of RNAs or proteins that influence different steps in the translation cycle • lox translation is regulated by the differentiation control element (DICE) sequence in the 3’ UTR and the hnRNP K and E1/ E2 proteins that bind it • These interactions prevent the large ribosomal subunit joining the small subunit during initiation • Drosophila the expression of protein Nanos is regulated by a 3’ UTR translation control elements (TCE) • the TCE sequence is bound by protein Smaug, which then interacts with cup (a 4E-BP) • This protein complex represses translation of Nanos by competing for normal eIF4G interactions • This mechanism is a variant of regulation of polyadenylation by the CPE element • micro RNAs (miRNAs) may also regulate translation of specific mRNAs in eukaryotes by binding partly complementary sequences in the 3’ UTR • miRNAs are thought to repress gene expression by interfering with translation initiation and coordinating with standard mRNA degradation. The molecular mechanisms of how initiation is down-regulated is still unknown Regulation of translation during elongation: phosphorylation of elongation factors • Translation can also be regulated downstream of initiation, including elongation, termination and recycling • For example, elongation factor eEF2 is phosphorylated in response to many forms of stress to globally regulate translation ◦When eEF2 is phosphorylated, it’s translocation promoting activity is greatly reduced ◦This leads to accumulation of pre-translocation-state ribosomes, which inhibits elongation • Elongation regulation often accompanies initiation regulation to help cells preserve and redirect limited resources under difficult conditions Regulation of translation during elongation: nascent peptide-mediated interactions • Both in bacteria and eukaryotes, nascent peptide can mediate stalling, regulating the translation of their coding mRNA • For example, for polycistronic mRNA in bacteria, ribosome stalling in the tnaC ORF is regulated by Tnac residues and tryptophan ◦The first ORF of operon tnaC contains a tryptophan-sensitive peptide stalling sequence • In low tryptophan, ribosomes complete synthesis of the tnaC ORF and are released, allowing the Rho terminator to bind RNA and terminate transcription • In high tryptophan, elongation stalls at a tryptophan-sensitive peptide stalling sequence, blocking Rho from binding via attached ribosome and transcription of downstream genes • The translated 24-aa TnaC peptide is important for this regulation: TnaC-stalled ribosome takes on a structure that is further stabilized by two free tryptophans binding to the TnaC peptide Viruses can hijack host translation machinery • Viral mRNAs use host machinery for their own translation, so they have developed ways to compete with their host for these resources, particularly in the initiation step • first, viruses can reduce translation of host mRNA by disrupting the host closed loop complex of mRNA ◦Picornaviruses (like polio) produce proteases that target eIF4G and PABP for cleavage ◦Flu viruses produce endonucleases that cleave host mRNA 5’ cap (but not viral caps) • Second, they can initiate translation specifically of viral mRNA. ◦Picornaviruses initiate translation of their mRNA through RNA elements called internal ribosome entry sites (IREs) on 5’ UTR. These large IREs RNA structures allow viral RNA to recruit small ribosomal subunits ◦IREs RNAs bind similar to where Shine-Dalgarno interactions would be located on bacterial ribosomes, thereby positioning the AUG start site at the P site ◦Picornaviruses then use their own factors - IREs-transacting factors (ITAFs) - to engage ribosome Viral Hijacking of the eIF2-dependent tRNA(met) loading • eIF2 is critical for loading the initiator tRNA(met) into their P site • However, phosphorylation of eIF2 influences the availability of eIF2:GTP • Host protein kinase RNA-activated (PKR) protein phosphorylates eIF2 when double-stranded RNA is encountered (similar to Gcn2 kinase during a.a. starvation), leading to a slow-down in translation • To avoid the PKR-mediated cellular response, viruses have developed different ways to initiate translation using IREs in their 5’ or 3’ UTRs to mimic the shape of a tRNA without the need of a start codon