The formation of intramolecular secondary structure brings mRNA ends in close proximity

Abstract

A number of protein factors regulate protein synthesis by bridging mRNA ends or untranslated regions (UTRs). Using experimental and computational approaches, we show that mRNAs from various organisms, including humans, have an intrinsic propensity to fold into structures in which the 5' end and 3' end are ≤ 7 nm apart irrespective of mRNA length. Computational estimates performed for ~22,000 human transcripts indicate that the inherent proximity of the ends is a universal property of most, if not all, mRNA sequences. Only specific RNA sequences, which have low sequence complexity and are devoid of guanosines, are unstructured and exhibit end-to-end distances expected for the random coil conformation of RNA. Our results suggest that the intrinsic proximity of mRNA ends may facilitate binding of translation factors that bridge mRNA 5' and 3' UTRs. Furthermore, our studies provide the basis for measuring, computing and manipulating end-to-end distances and secondary structure in mRNAs in research and biotechnology. INRODUCTIONRegulation of mRNA translation in eukaryotes involves protein-mediated interactions between mRNA ends. Translation initiation requires the recruitment of the small ribosomal subunit to the 5' end of the mRNA 1 . The formation of the initiation complex is stimulated by the interaction between the 5' mRNA cap-binding protein eIF4E and the 3' end poly(A) tail binding protein PABP, which is mediated through their binding to different parts of the translational factor eIF4G 2,3 . The eIF4E•eIF4G•PABP complex is thought to enhance translation initiation by circularizing the mRNA and forming the "closed-loop" structure 4-6 . The mechanism by which the mRNA closed loop enhances proteins synthesis is not well understood.Remarkably, translation initiation of many eukaryotic mRNAs is also regulated by sequences in their 3' UTRs and controlled by the formation of protein bridges between the 5' and 3' UTRs. For example, the 3' UTR regulatory sequences recruit protein complexes (e.g. CPEB•Maskin, Bruno•Cup, or GAIT complex), which inhibit translation by interacting with either eIF4E or eIF4E•eIF4G bound to the 5' end of mRNA 7 . The pervasiveness of protein bridges between mRNA UTRs in the evolution of translation regulation is puzzling because of the significant entropic cost expected for protein-mediated mRNA circularization 8 .The entropic penalty for the formation of protein bridges between mRNA ends may be partially mitigated by mRNA compaction through intramolecular basepairing interactions.Recent theoretical analyses suggested that the 5' and 3' ends of long (1,000-10,000 nucleotidelong) RNAs are always brought in the proximity of few nanometers of each other regardless of RNA length and sequence because of the intrinsic propensity of RNA to form widespread intramolecular basepairing interactions 8-10 . One study predicted that the 5' to 3' end distance in RNAs is 3 nm, on average 8 . These theoretical predictions were tested by single-molecule Förster resonance energy transfer (smFRET) measurements o...

mRNP architecture in translating and stress conditions reveals an ordered pathway of mRNP compaction

“…Several computation studies suggest that the ends of mRNAs are close (<10 nm) for RNAs in solution (Fang et al, 2011; Yoffe et al, 2011; Clote et al, 2012). Moreover, an in vitro FRET-based assay indicates that for all 11 mRNAs examined, the distance between the 5′ and 3′ ends is <10 nm (Lai et al, 2018 Preprint ). This distance is significantly smaller than one would expect if it these RNAs were behaving as a random coil in solution (Lai et al, 2018 Preprint ).…”

“…Moreover, an in vitro FRET-based assay indicates that for all 11 mRNAs examined, the distance between the 5′ and 3′ ends is <10 nm (Lai et al, 2018 Preprint ). This distance is significantly smaller than one would expect if it these RNAs were behaving as a random coil in solution (Lai et al, 2018 Preprint ). Therefore, under stress conditions, if most mRNAs are now exposed and can form significant intramolecular interactions, the properties of RNA as a polymer might promote the interaction of the 5′ and 3′ ends.…”

Revisiting the Closed-Loop Model and the Nature of mRNA 5′–3′ Communication

“…100% corresponds to base pairing between the 5′-most and the 3′-most nucleotides. Many 1000– 6000-nt long mRNAs from viruses and eukaryotes have a relative 5′–3′ proximity >98.5% (Lai et al, 2018; Leija-Martinez et al, 2014). A striking example is the 18,431 nt long MDN1 mRNA, for which a 136 nm median distance between the 5′ and 3′ ends (only 7 times larger than the diameter of a eukaryotic ribosome) is ~45-fold smaller than the distance within an extended coil conformation (>6 μm); upon removal of ribosomes, the MDN1 mRNA further compacts so that the median distance is 36 nm (Adivarahan et al, 2018).…”

mRNP architecture in translating and stress conditions reveals an ordered pathway of mRNP compaction

“…One hypothesis is that the closed-loop conformation is a non-stable 363 state during translation and that in the absence of translation, the closed loop confirmation can 364 form through interactions of eIF4E, eIF4G, and PABP where eIF4E binds to the m 7 G cap, 365 PABP binds to poly(A) tail, and eIF4G binds to both eIF4E and PABP (Hinnebusch and Lorsch,366 2012) ( Figure 6). Alternatively, or in addition, the 5' and 3' ends of mRNPs may be close during 367 stress because of an intrinsic property of "naked" RNAs to fold in a manner that brings the ends 368 FRET-based assay indicates for all eleven mRNAs examined, the distance between the 5' and 3' 371 ends are less than 10 nm (Lai et al, 2018). This distance is significantly smaller than one would 372 expect if it these RNAs were behaving as a random coil in solution (Lai et al, 2018).…”

“…Alternatively, or in addition, the 5' and 3' ends of mRNPs may be close during 367 stress because of an intrinsic property of "naked" RNAs to fold in a manner that brings the ends 368 FRET-based assay indicates for all eleven mRNAs examined, the distance between the 5' and 3' 371 ends are less than 10 nm (Lai et al, 2018). This distance is significantly smaller than one would 372 expect if it these RNAs were behaving as a random coil in solution (Lai et al, 2018). Therefore, 373 under stress conditions, if most mRNAs are now exposed and can form significant 374 intramolecular interactions, its properties as a polymer might promote the interaction of the 5' 375 and 3' ends.…”