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Telomeric repeat-containing RNA is a new noncoding RNA molecule that performs various biofunctions. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an RNA-binding protein involved in the telomere maintenance machinery. To date, little is known about how hnRNPA1 binds to telomeric RNA. In this study, we investigated the binding affinity and recognition mechanism of telomere RNA with the RNA recognition motif of hnRNPA1. Using the photochemical cross-linking method, we showed that the telomere RNA G-quadruplex with loops is important in the interaction of telomere RNA with hnRNPA1. Using small-molecule probes, we directly visualized the complex formed by the telomere RNA G-quadruplex and hnRNPA1 in vitro and in live cells. The results suggested that the structure-dependent binding of hnRNPA1 to telomere RNA regulates the telomere function. Therefore, our study provides new insights into the interactions between the RNA G-quadruplex and proteins at the telomere.

Human telomeric RNA performs various cellular functions such as telomere length regulation, heterochromatin formation, and chromosome end protection. Using a combination of nuclear magnetic resonance, circular dichroism, and gel electrophoresis, we observed an unusual topological structure formed by human telomere RNA r(GUUAGGGU). Our results showed that every set of four strands formed a parallel G-quadruplex as symmetry-related units containing four G-tetrads, two U-tetrads, and one A-tetrad. An eight-stranded helical fragment containing A-, G-, and U-tetrads provided a central intercalated scaffold that connected two G-quadruplex units in an alternating antiparallel arrangement, giving rise to a novel RNA architecture. This higher order RNA structure is so stable that it would be surprising if similar structures do not occur in nature. Our findings provide a new insight into the behavior of human telomeric RNA molecules.

The understanding of telomeres is expected to provide major insights into genome stability, cancer, and telomere-related diseases. In recent years, there have been considerable improvements in the technologies available to determine the length of telomeres of human chromosomes; however, the present methods for measuring telomere length are fraught with shortcomings that have limited their use. Here we describe a method for detection of individual telomere lengths (DITL) that uses a chemistry-based approach that accurately measures the telomere lengths from individual chromosomes. The method was successfully used to determine telomere DNA by breaking in the target sequence and producing a "real telomere fragment." The DITL approach involves cleavage of the sequence adjacent to the telomere followed by resolution of the telomere length at the nucleotide level of a single chromosome. Comparison of the DITL method and the traditional terminal restriction fragment (TRF) analysis indicates that the DITL approach appears to be promising for the quantification of telomere repeats in each chromosome and the detection of accurate telomere lengths that can be missed using TRF analysis.

Telomeric repeat-containing RNA is a new noncoding RNA molecule recently discovered in mammalian cells. Here we report the structural features of human telomere RNA r(UAGGGU) in the presence of K(+) and Na(+). We demonstrated for the first time that a novel U-tetrad is formed at the 3' end of a parallel human telomeric RNA G-quadruplex. The U-tetrad dramatically stabilizes human telomeric RNA G-quadruplex structure, leading to an increase in melting temperature (T(m)) of 29 degrees C. The U-tetrad-stabilized telomeric RNA G-quadruplex structure adds considerably to our understanding of the diversity of RNA G-quadruplex architectures. It shows that the structure of base "quartets" is important in RNA assembly. The structural information will be invaluable for understanding the function of human telomere RNA.