Structural Diversity of Triplet Repeat RNAs

Abstract

Tandem repeats of various trinucleotide motifs are present in the human transcriptome, but the functions of these regular sequences, which likely depend on the structures they form, are still poorly understood. To gain new insight into the structural and functional properties of triplet repeats in RNA, we have performed a biochemical structural analysis of the complete set of triplet repeat transcripts, each composed of a single sequence repeated 17 times. We show that these transcripts fall into four structural classes. The repeated CAA, UUG, AAG, CUU, CCU, CCA, and UAA motifs did not form any higher order structure under any analyzed conditions. The CAU, CUA, UUA, AUG, and UAG repeats are ordered according to their increasing tendency to form semistable hairpins. The repeated CGA, CGU, and all CNG motifs form fairly stable hairpins, whereas AGG and UGG repeats fold into stable G-quadruplexes. The triplet repeats that formed the most stable structures were characterized further by biophysical methods. UV-monitored structure melting revealed that CGG and CCG repeats form, respectively, the most and least stable hairpins of all CNG repeats. Circular dichroism spectra showed that the AGG and UGG repeat quadruplexes are formed by parallel RNA strands. Furthermore, we demonstrated that the different susceptibility of various triplet repeat transcripts to serum nucleases can be explained by the sequence and structural features of the tested RNAs. The results of this study provide a comprehensive structural foundation for the functional analysis of triplet repeats in transcripts.Trinucleotide repeats (TNRs) 4 belong to a family of microsatellite sequences also known as short tandem repeats or simple sequence repeats. TNRs are present in both prokaryotic and eukaryotic genomes and, similar to other microsatellites, show high mutation rates resulting in frequent length variability. Polymorphic TNRs are better tolerated than dinucleotide and tetranucleotide repeats in translated sequences because their length variation does not change the open reading frame.A recent survey of the human genome reference sequence showed that it harbors more than 32,000 tracts of uninterrupted TNR sequences composed of six or more repeated units. In annotated human exons, which account for less than 3% of the genomic sequence, there are as many as 1,030 TNR tracts (61). Some AT-rich TNR types, such as CTT, AAC, and AAT, are particularly underrepresented in exons, whereas GC-rich repeats (CGG, CAG, and CCG) are highly overrepresented, implying that these sequences have a functional significance. The AT-rich and GC-rich TNRs tend to localize preferentially in the 3Ј-UTR and 5Ј-UTR, respectively (1). About 60% of exonic TNRs are localized in the open reading frame. These coding repeats are primarily translated to the poly-Gln, polyAla, poly-Glu, and poly-Leu tracts. However, the amino acid coding property of TNRs is not the only feature for which these sequences are selected in exons. The other properties of TNR sequences that manifest thems...

The Disease-associated r(GGGGCC) Repeat from the C9orf72 Gene Forms Tract Length-dependent Uni- and Multimolecular RNA G-quadruplex Structures

“…Circular Dichroism Shows That the r(GGGGCC) n Repeat Forms a Parallel G-quadruplex-CD spectroscopy is a biophysical means to assess G-quadruplex formation in RNA (28,29). CD analysis of the r(GGGGCC) 4 repeat in potassium buffer revealed a positive peak around 260 nm and a negative peak around 240 nm (Fig.…”

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Triplet repeats in transcripts: structural insights into RNA toxicity

“…The cleavage patterns of these transcripts determined at 37 ° C do not show any hallmarks of hairpin structures. Finally, as many as seven TNR RNAs, the AAG, CAA, CCA, CCU, CUU, UAA and UUG motifs, appeared unable to form any secondary structures (Sobczak et al , 2010 ). The structural variety of the TNR RNAs may reflect their potential to interact with a variety of RNA binding proteins and perform diverse biological functions.…”

“…However, this number is reduced to 20 different TNRs when the four homonucleotide motifs are excluded and the three different phases of the 60 individual sequence motifs are combined. Recently, the ability of these TNRs to adopt higher-order RNA structures has been investigated by chemical and enzymatic structure probing and biophysical methods (Sobczak et al , 2010 ). As a result, the TNRs have been divided into four structural classes: (I) very stable G-quadruplex structures, known also as G-quartets; (II) semi-stable hairpins; (III) unstable hairpins; and (IV) not forming any higher order structures (Figure 1 A).…”