Iron regulatory protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs.

Abstract

Communicated by W.I.MattajTranslation of ferritin and erythroid 5-aminolevulinate synthase (eALAS) mRNAs is regulated by iron via mRNA-protein interactions between iron-responsive elements (IREs) and iron regulatory protein (IRP). In iron-depleted cells, IRP binds to single IREs located in the 5' untranslated regions of ferritin and eALAS mRNAs and represses translation initiation. The molecular mechanism underlying this translational repression was investigated using reconstituted, IRE-IRP-regulated, cell-free translation systems. The IRE-IRP interaction is shown to prevent the association of the 43S translation pre-initiation complex (including the small ribosomal subunit) with the mRNA. Studies with the spliceosomal protein UlA and mRNAs which harbour specific binding sites for this protein in place of an IRE furthermore reveal that the 5' termini of mRNAs are generally sensitive to repressor proteinmediated inhibition of 43S pre-initiation complex binding.

Ribosomal Pausing and Scanning Arrest as Mechanisms of Translational Regulation from Cap-Distal Iron-Responsive Elements

“…Plasmids NOP1 and IRE-mut have been previously described (11,41). IRE.34 was created by insertion of a BamHI-XbaI fragment containing the IRE from F64 (10) into IRE-wt (11,13) digested with BamHI-XbaI. IRE.66 was created from IRE-wt by digestion with BamHI and filling of the BamHI cohesive ends by using Klenow fragment, prior to digestion with XbaI and insertion of a StuI fragment from F64 containing the IRE and sequences 5Ј of the IRE.…”

“…Translation initiation assays and sucrose gradient analysis were performed as described previously (12,13).…”

“…Translational control by specific mRNAprotein interactions is commonly enacted at the level of translation initiation (e.g., caudal [6,38], 15-lipoxygenase [35,36], and oskar [22,43]). IRP binding to the IRE of ferritin mRNAs affects an early step of translation initiation: it prevents the recruitment of the 43S translation preinitiation complex (which includes the small ribosomal subunit) (13,33). Transfection studies using mammalian tissue culture cells revealed a characteristic feature of this IRE-IRP regulatory mechanism: for IRP binding to efficiently block translation, the IRE must be located within Ͻ60 nucleotides from the m 7 GpppN-cap structure of the mRNA (9,10).…”

A stable hairpin preceded by a short open reading frame promotes nonlinear ribosome migration on a synthetic mRNA leader

“…In eukaryotic cells, translation initiation usually occurs according to the ribosome scanning mechanism (Kozak, 1989b)+ This classical model assumes that, at first, the 40S preinitiation complex associates with an mRNA at the 59-terminal cap structure and later unidirectionally scans the mRNA leader until the first AUG codon positioned in an appropriate context is encountered, whereupon the 60S subunit joins and protein synthesis starts+ Translation can be strongly affected by cis-acting elements and trans-acting factors that interfere with ribosome scanning in the leader: (1) highly stable stemloop structures mechanically block scanning by stalling 40S subunits on the 59 site of the hairpin (Pelletier & Sonenberg, 1985;Kozak, 1986Kozak, , 1989a; (2) upstream open reading frames (uORFs) reduce initiation downstream due to the apparent low efficiency of reinitiation at subsequentAUG codons (Kozak, 1989b(Kozak, , 1992Hinnenbusch, 1994); (3) RNA-binding proteins associated with cap-proximal binding sites inhibit recognition of the cap structure due to the steric hindrance (Gray & Hentze, 1994;Stripecke et al+, 1994)+ Modified ribosome scanning mechanisms exist, however, that allow relatively efficient translation despite the presence of inhibitory elements in the leader+ Alternative modes of translation initiation have been broadly studied and fall into several categories: leaky scanning (Kozak, 1992), reinitiation (Geballe & Morris, 1994;Hinnenbusch, 1994), internal initiation (Pelletier & Sonenberg, 1988;Kaminski et al+, 1990;Belsham, 1992), and nonlinear ribosome scanning or ribosome shunt (Curran & Kolakofsky, 1988, 1989Fütterer et al+, 1989Fütterer et al+, , 1993Fütterer et al+, , 1996Yueh & Schneider, 1996)+ The cauliflower mosaic virus (CaMV) pregenomic 35S RNA 600-nt leader (Figs+ 1 and 2A) contains all three elements with the potential to strongly inhibit translation: (1) a low-energy elongated hairpin (Ϫ110 kcal/ mol) that has been characterized by enzymatic and chemical probing (Hemmings-Mieszczak et al+, 1997), (2) several uORFs, and (3) a purine-rich cloverleaf structure that interacts with the CaMV coat protein (O+ Guerra-Peraza, M+ de Tapia, T+ Hohn, and M+ HemmingsMieszczak, submitted)+ However, translation initiation downstream of the leader is cap dep...…”

A polysomal ribonuclease involved in the destabilization of albumin mRNA is a novel member of the peroxidase gene family

“…Western blot analysis of PMR-1 following estrogen stimulation+ Ten microgram samples of hepatocyte protein isolated at the indicated times following estrogen stimulation were analyzed by Western blot using a polyclonal antiserum to PMR-1+ Lane 1 is a standard of 1 ng of purified PMR-1+ A parallel blot of 100 ng of hepatocyte protein per lane was probed with sheep antiserum to vitellogenin as a control for estrogen stimulation+ ern blot in Figure 1 provide suggestive evidence that, like hMPO, PMR-1 is made as a larger precursor and is proteolytically processed to the form identified on polysomes+ It may or may not be a coincidence that the site of this putative processing event occurs at a position similar to that which separates the propeptide and light chain of hMPO+ Expression of the full-length PMR-1 cDNA in a recombinant baculovirus (Fig+ 4) yielded the 80-kDa protein (PMR80) expected from the sequence, but this was not processed to the 60-kDa form isolated from liver polysomes and lacked enzymatic activity+ These data indicate both that Sf9 cells lack the enzymatic pathway by which PMR-1 is proteolytically processed, and that processing is required to generate a catalytically active form of the enzyme+ In contrast, the recombinant 60-kDa form of PMR-1 estimated to represent the protein isolated from polysomes (PMR60) is an active endonuclease (Figs+ 5-7)+ PMR-1 was originally identified by its ability to generate a doublet cleavage product from a portion of the 59 end of albumin mRNA (Pastori et al+, 1991b)+ This was later mapped to consensus overlapping APyrUGA elements in a singlestranded loop region (Chernokalskaya et al+, 1997)+ That study also demonstrated that the purified enzyme also cleaved albumin mRNA at a number of sites that did not correspond to this consensus sequence+ Recombinant PMR60 did not cleave albumin mRNA at consensus APyrUGA elements (Fig+ 7)+ However, the recombinant enzyme generated much the same pattern of nonconsensus cleavages as the purified enzyme, indicating both that it is an endonuclease and that it is indeed PMR-1+ The reason recombinant PMR60 does not cleave at APyrUGA elements is not known, although we suspect it is related to folding of the protein, because a similar result was obtained with purified PMR-1 that was renatured after treatment under denaturing conditions (data not shown)+ It is unclear how a putative messenger RNase evolved from the peroxidase gene family+ Myeloperoxidase lacks detectable ribonuclease activity (Fig+ 2C), indicating this family of proteins does not have a secondary enzymatic function+ Perhaps some insight into this may be found in the dual function of several metabolic enzymes as RNA-binding proteins+ IRP-1 functions as a regulator of iron metabolism through its role in the translational repression of ferritin mRNA (Gray & Hentze, 1994) and stabilization of transferrin receptor mRNA (Binder et al+, 1994)+ Iron binding to the active site of this protein converts IRP1 from its RNA-binding form to cytoplasmic aconitase (Hirling et al+, 1994)+ Glyceraldehyde-3-phosphosphate dehydrogenase is a well-characterized hydrolytic enzyme which can bind AU-rich RNA sequence elements through its NAD(ϩ) binding site (Nagy & Rigby, 1995)+ Glutamate dehydrogenase was identified as the protein whose binding to the 39 UTR of cytochrome c oxidase mRNA correlates with liver-specific mRNA stabilization (Preiss et al+, 1995), and AUH, an ARE-binding protein, has enol...…”