Structure of 18 S Ribosomal RNA in Native 40 S Ribosomal Subunits

Abstract

We have analyzed the structure of 18 S rRNA in native 40 S subunits using chemical modification followed by primer extension. The native subunits were modified using the single-stranded specific reagents dimethyl sulfate and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate. The modification pattern of the 18 S rRNA was compared to that obtained from 1 binds to 40 S subunits and prevents formation of unprogrammed 80 S ribosomes by inhibiting association of the 40 S and 60 S ribosomal subunits in the absence of mRNA. Initiation factor eIF-2 selects the specific initiator tRNA (MettRNA f ) and brings it to the 40 S subunit. The resulting 43 S pre-initiation complex binds mRNA with the help of a series of initiation factors. The 60 S subunit now joins the mRNA containing 48 S initiation complex in a reaction that requires an additional initiation factor (eIF-5) and is associated with the hydrolysis of GTP.Several of the initiation factors are found to be associated with the so-called native 40 S ribosomal subunits (40 S N ) in vivo (2). Most of these factors are present on the 40 S N particles in small quantities, but eIF-3 is present in stoichiometric amounts (2). Initiation factor 3 is a huge multisubunit protein with a total mass of approximately 0.7 MDa (3). The factor displays RNA binding properties, and one of its subunits can be cross-linked to 18 S rRNA in the 40 S⅐eIF-3 complex (4). This suggests that rRNA may, at least in part, be responsible for binding the factor to the small ribosomal subunit. However, the location of the eIF-3 interaction site in 18 S rRNA is not known.The ribosomal RNA is considered to be involved in various ribosomal functions such as A-and P-site-related activities and peptide bond formation (for a review see Ref. 5). In prokaryotes the rRNA is directly involved in the binding of initiation factors and mRNA during protein synthesis initiation (6 -9). Less is known about the functional role of rRNA in the eukaryotic ribosome, but studies using chemical cross-linking and chemical and enzymatic footprinting have indicated that the rRNA is involved in mRNA binding, subunit interaction, and binding of elongation factors (10 -12).We have previously studied the structure of 18 S rRNA in derived 40 S subunits prepared by dissociation of isolated 80 S ribosomes (11, 13). In contrast to the native subunits, derived particles are free from additional non-ribosomal proteins. In this report, we have compared the structures of 18 S rRNA in native and derived 40 S subunits using chemical modification. The two types of 18 S rRNAs showed distinct but limited structural differences. The role of the non-ribosomal proteins in altering the structure of the 18 S rRNA in the 40 S N particles is discussed. Nygård and Nika (14). Briefly, isolated monosomes (15) were suspended in 0.5 M KCl, 20 mM Tris/HCl, pH 7.6, 3 mM MgCl 2 , and 10 mM 2-mercaptoethanol. The material was layered onto continuous 10 -40% (w/v) sucrose gradients containing 0.35 M KCl, 20 mM Tris/HCl, pH 7.6, 3 mM MgCl ...

Genome-wide profiling of mouse RNA secondary structures reveals key features of the mammalian transcriptome

“…Our data are in agreement with previous reports showing DMS reactivity with G/U residues [36,45-47] and CMCT reactivity with cytosines [48-50]. We observed a significant increase in the accuracy of the de novo prediction of structures when considering also these non-canonical reactivities as both the canonical and non-canonical reactivities lay within single-stranded regions (Figure S3C in Additional file 1).…”

Genome-wide profiling of mouse RNA secondary structures reveals key features of the mammalian transcriptome

“…Our data are in agreement with previous reports showing DMS reactivity with G/U residues [36,[45][46][47] and CMCT reactivity with cytosines [48][49][50]. We observed a significant increase in the accuracy of the de novo prediction of structures when considering also these non-canonical reactivities as both the canonical and non-canonical reactivities lay within single-stranded regions ( Figure S3C in Additional file 1).…”

The UV-inducible RNA-binding Protein A18 (A18 hnRNP) Plays a Protective Role in the Genotoxic Stress Response

“…Based on sequence analysis we have determined that the 18 S rRNA sequence bound to A18 hnRNP corresponds to nucleotides 706 -1385 of the 18 S rRNA (data not shown). This sequence is located in the central domain of the 18 S rRNA and is thought to be involved in eIF3 interaction, which prevents premature association of the large and small ribosomal subunits (25). A potential role in translation regulation is also supported by the binding of A18 hnRNP to the transcripts of two translational elongation factors (1␣ and 1␤2).…”