Human intron-encoded AluACA RNAs and telomerase RNA share a common element promoting RNA accumulation

Mammalian cells express hundreds of intron-encoded box H/ACA RNAs which fold into a common hairpin-hinge-hairpin-tail structure, interact with 4 evolutionarily conserved proteins, dyskerin, Nop10, Nhp2 and Gar1, and function mainly in RNA pseudouridylation. The human telomerase H/ACA RNA (hTR) directs telomeric DNA synthesis and it carries a 5-terminal domain encompassing the telomeric template sequence. The primary hTR transcript is synthesized from an independent gene by RNA polymerase II and undergoes 3 end processing controlled by the 3-terminal H/ACA domain. The apical stem-loop of the 3 hairpin of hTR carries a unique biogenesis-promoting element, the BIO motif that promotes hTR processing and RNP assembly. AluACA RNAs represent a distinct class of human H/ACA RNAs; they are processed from intronic Alu repetitive sequences. As compared to canonical H/ACA RNAs, the AluACA RNAs carry unusually short or long 5 hairpins and generally, they accumulate at low levels. Here, we demonstrate that the suboptimal 5 hairpins are responsible for the weak expression of AluACA RNAs. We also show that AluACA RNAs frequently carry a processing/stabilization element that is structurally and functionally indistinguishable from the hTR BIO motif. Both hTR and AluACA biogenesis-promoting elements are located in the terminal stem-loop of the 3-terminal H/ACA hairpin, they show perfect structural conservation and are functionally interchangeable in in vivo RNA processing reactions. Our results demonstrate that the BIO motif, instead of being confined to hTR, is a more general H/ACA RNP biogenesis-facilitating element that can also promote processing/assembly of intron-encoded AluACA RNPs

Dynamic association of human mRNP proteins with mitochondrial tRNAs in the cytosol

Cytoplasmic localization, stability, and translation of mRNAs are controlled by their dynamic association of numerous mRNA-binding (mRNP) proteins, including cold shock domain (CSD)-containing proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), and serine/arginine-rich (SR) proteins. Here, we demonstrate that the most abundant human mRNP protein, the CSD-containing Y-box-binding protein 1 (YBX1), the closely related YBX3 protein, and other mRNP proteins, such as SRSF1, SRSF2, SRSF3, hnRNP Al, and H, specifically and efficiently interact with overlapping sets of mitochondrial tRNAs (mt tRNAs). In vitro reconstitution and in vivo binding experiments show that YBX1 recognizes the D- and/or T-stem-loop regions of mt tRNAs through relying on the RNA-binding capacity of its CSD. Cell fractionation and in vivo RNA-protein cross-linking experiments demonstrate that YBX1 and YBX3 interact with mt tRNAs in the cytosol outside of mitochondria. Cell fractionation and fluorescence in situ hybridization experiments provide evidence that mitochondrial autophagy promotes the release of mt tRNAs from the mitochondria into the cytoplasm. Association of mRNP proteins with mt tRNAs is highly dynamic; it is rapidly increased upon transcription inhibition and decreased during apoptosis. Although the cytoplasmic function of mt tRNAs remains elusive, their dynamic interactions with key mRNA-binding proteins may influence cytoplasmic mRNA stability and/or translation

Guide RNA acrobatics: positioning consecutive uridines for pseudouridylation by H/ACA pseudouridylation loops with dual guide capacity

International audienceSite-specific pseudouridylation of human ribosomal and spliceosomal RNAs is directed by H/ACA guide RNAs composed of two hairpins carrying internal pseudouridylation guide loops. The distal “antisense” sequences of the pseudouridylation loop base-pair with the target RNA to position two unpaired target nucleotides 5′-UN-3′, including the 5′ substrate U, under the base of the distal stem topping the guide loop. Therefore, each pseudouridylation loop is expected to direct synthesis of a single pseudouridine (Ψ) in the target sequence. However, in this study, genetic depletion and restoration and RNA mutational analyses demonstrate that at least four human H/ACA RNAs (SNORA53, SNORA57, SCARNA8, and SCARNA1) carry pseudouridylation loops supporting efficient and specific synthesis of two consecutive pseudouridines (ΨΨ or ΨNΨ) in the 28S (Ψ3747/Ψ3749), 18S (Ψ1045/Ψ1046), and U2 (Ψ43/Ψ44 and Ψ89/Ψ91) RNAs, respectively. In order to position two substrate Us for pseudouridylation, the dual guide loops form alternative base-pairing interactions with their target RNAs. This remarkable structural flexibility of dual pseudouridylation loops provides an unexpected versatility for RNA-directed pseudouridylation without compromising its efficiency and accuracy. Besides supporting synthesis of at least 6% of human ribosomal and spliceosomal Ψs, evidence indicates that dual pseudouridylation loops also participate in pseudouridylation of yeast and archaeal rRNAs

Frequent mutations of the CA simple sequence repeat in intron 1 of EGFR in mismatch repair-deficient colorectal cancers

AIM: To investigate the polymorphic simple sequence repeat in intron 1 of the epidermal growth factor receptor gene (EGFR) (CA-SSRI), which is known to affect the efficiency of gene transcription as a putative target of the mismatch repair (MMR) machinery in colorectal tumors