Initiation of Translation of the FMR1 mRNA Occurs Predominantly through 5′-End-Dependent Ribosomal Scanning

Abstract

The fragile X mental retardation 1 (FMR1) gene contains a CGG repeat within its 5′ untranslated region (5′UTR) that, when expanded to 55–200 CGG repeats (premutation allele), can result in the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome. The CGG repeat is expected to form a highly stable secondary structure that is capable of inhibiting 5′-cap-dependent translation. Paradoxically, translation in vivo is only mildly impaired within the premutation range, suggesting that other modes of translation initiation may be operating. To address this issue, we translated in vitro a set of reporter mRNAs containing between 0 and 99 CGG repeats in either native (FMR1) or unrelated (heterologous) 5′UTR context. The 5′-cap dependence of translation was assessed by inserting a stable hairpin (HP) near the 5′ end of the mRNAs. The results of the current studies indicate that translation initiation of the FMR1 mRNA occurs primarily by scanning, with little evidence of internal ribosome entry or shunting. Additionally, the efficiency of translation initiation depends on transcription start site selection, with the shorter 5′UTR (downstream transcription start site I) translating with greater efficiency compared to the longer mRNA (start site III) for all CGG-repeat elements studied. Lastly, an HP previously shown to block translation gave differing results depending on the 5′UTR context, in one case initiating translation from within the HP.

Introduction

The 5′ untranslated region (5′UTR) of the human fragile X mental retardation 1 (FMR1) gene (Online Mendelian Inheritance in Man ID* 309550) harbors a CGG repeat that may expand generationally.1, 2 Whereas the general population has fewer than 45 CGG repeats (mode, ∼ 30 CGG repeats), full-mutation allelic expansions (> 200 CGG repeats) are normally accompanied by FMR1 gene silencing3, 4, 5, 6 and loss of FMR1 protein (FMRP).7, 8, 9 This absence of FMRP gives rise to fragile X syndrome, the most common known heritable form of intellectual impairment and leading single-gene form of autism (for reviews, see Bassell and Warren⁸ and Hagerman et al.¹⁰). Smaller “premutation” expansions (55–200 CGG repeats) are also associated with increased risk of developmental delay and autism.11, 12 Premutation expansions are additionally linked to two premutation-specific disorders: primary ovarian insufficiency (with loss of ovarian function before the age of 40 years)13, 14, 15, 16 and the neurodegenerative disorder fragile X-associated tremor/ataxia syndrome,17, 18, 19 which involves the core features of intention tremor and gait ataxia and, more variably, cognitive decline, parkinsonism, and peripheral neuropathy (reviewed by Berry-Kravis et al.²⁰ and Leehey²¹). In contrast to full-mutation alleles, which are generally transcriptionally silent, premutation alleles express up to eight times more FMR1 mRNA than normal alleles.22, 23, 24, 25, 26 Although the mechanism is not well understood, fragile X-associated tremor/ataxia syndrome—perhaps primary ovarian insufficiency as well—is thought to be due to a toxic gain-of-function of the expanded CGG-repeat mRNA.17, 27, 28, 29

The CGG repeat is representative of a growing number of known trinucleotide repeat disorders that include both noncoding repeats (e.g., CTG, myotonic dystrophy; GAA, Friedreich's ataxia; CGG, fragile X syndrome) and protein-coding repeats (e.g., CAG, Huntington's disease, and spinocerebellar ataxias).30, 31 Because the CGG repeat of the FMR1 mRNA lies outside of the coding region, it does not have a direct effect on the composition of FMRP; however, the location of this structure-forming CG-rich element in the 5′UTR of the mRNA directly affects the efficiency of FMRP production.

Ribo-CGG repeats have been shown experimentally to base-pair intramolecularly and to form hairpin (HP)-like secondary structures of C-G and G-G base pairs in vitro.32, 33, 34, 35 Some investigators have also reported tetraplex formation by CGG-repeat RNA.36, 37 This highly GC-rich secondary structure is thought to inhibit the translation of expanded-repeat FMR1 mRNAs, since a strong secondary structure in the 5′UTR can inhibit ribosomal scanning.38, 39, 40 Secondary structures with free energies of stabilization more negative than approximately − 50 kcal/mol greatly inhibit translation initiation.39, 41 By comparison, CGG repeats at the lower end of the premutation range have estimated free energies below − 100 kcal/mol; a repeat of 55 CGGs has an in silico estimated free energy of stabilization of − 117 kcal/mol.42, 43 Therefore, CGG repeats are predicted to pose a substantial energetic barrier for the scanning of the FMR1 5′UTR by the 40S ribosome and associated proteins. In addition to the repeat tract, the FMR1 5′UTR otherwise is relatively long (198 bases) and GC-rich (77%).

Paradoxically, patients with mRNAs carrying 50–100 CGG repeats have only slightly reduced levels of FMRP.22, 26, 44, 45 Furthermore, a polysome profile analysis of lymphoblastoid cells carrying 97 CGG repeats shows a substantial amount (58%) of FMR1 mRNA in polysomes, suggesting maintenance of at least a moderate rate of protein synthesis.⁴⁴ In this regard, transient transfections of mammalian cells with plasmids containing 99 CGG repeats resulted in only an ∼ 50% loss in reporter translation efficiency relative to 30 CGG-repeat constructs.46, 47 Thus, it remains a fundamental puzzle as to why FMR1 messages in the premutation range can be translated while harboring substantial 5′UTR secondary structures.

It is possible that canonical “ribosomal scanning” (reviewed by Pestova et al.⁴⁸) is not operational for the translation initiation of the FMR1 mRNA; alternative models of translation initiation have been developed in order to interpret unusual forms of initiation that are not compatible with scanning. For example, higher-order RNA structures within internal ribosome entry sites (IRESs) are thought to have an intrinsic ability to directly bind the 40S ribosome, at or near a transcript's AUG start codon, without 5′-m7G cap binding or ribosomal scanning.⁴⁹ Often seen in viral50, 51 as well as cell cycle and apoptotic messages,52, 53 IRES elements facilitate protein synthesis in a 5′-cap-independent manner.

Another alternative to ribosomal scanning is ribosomal shunting, a rare mechanism of initiation that is primarily seen in plant viruses.54, 55 Although shunting does require 5′-cap binding by ribosomal initiation factors, the ribosome is able to bypass large and highly structured RNA domains within the 5′UTR, usually by way of initiation at upstream open reading frames, followed by reinitiation downstream of the structured domain. Since the FMR1 5′UTR is long and highly structured, especially messages with premutation-length CGG repeats, this alternative form of translation initiation in principle could allow for a more efficient translation of FMR1.

To address the issue of translation initiation for the FMR1 mRNA, we have examined the above alternative mechanisms. Our results confirm earlier observations22, 44, 47, 56 that CGG repeats inhibit translation initiation in a length-dependent manner. Moreover, when the CGG repeat is placed in an unrelated (heterologous) 5′UTR context, a similar effect is seen. Replacing the CGG repeat with a double-stranded HP completely blocks translation in a heterologous 5′UTR, whereas the same HP decreases by about 5-fold but does not abolish translation in the FMR1 context. We provide evidence that translation of FMR1 is 5′-cap-dependent, does not substantially involve IRES-mediated initiation, and most likely occurs by ribosomal scanning. We also give possible explanations for HP read-through in the FMR1 context.