Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Sep 1;26(17):1897-910.
doi: 10.1101/gad.197467.112. Epub 2012 Aug 14.

Human intron-encoded Alu RNAs are processed and packaged into Wdr79-associated nucleoplasmic box H/ACA RNPs

Beáta E Jády  1 , Amandine Ketele, Tamás Kiss
Affiliations

Human intron-encoded Alu RNAs are processed and packaged into Wdr79-associated nucleoplasmic box H/ACA RNPs

Beáta E Jády et al. Genes Dev. .

Abstract

Alu repetitive sequences are the most abundant short interspersed DNA elements in the human genome. Full-length Alu elements are composed of two tandem sequence monomers, the left and right Alu arms, both derived from the 7SL signal recognition particle RNA. Since Alu elements are common in protein-coding genes, they are frequently transcribed into pre-mRNAs. Here, we demonstrate that the right arms of nascent Alu transcripts synthesized within pre-mRNA introns are processed into metabolically stable small RNAs. The intron-encoded Alu RNAs, termed AluACA RNAs, are structurally highly reminiscent of box H/ACA small Cajal body (CB) RNAs (scaRNAs). They are composed of two hairpin units followed by the essential H (AnAnnA) and ACA box motifs. The mature AluACA RNAs associate with the four H/ACA core proteins: dyskerin, Nop10, Nhp2, and Gar1. Moreover, the 3' hairpin of AluACA RNAs carries two closely spaced CB localization motifs, CAB boxes (UGAG), which bind Wdr79 in a cumulative fashion. In contrast to canonical H/ACA scaRNPs, which concentrate in CBs, the AluACA RNPs accumulate in the nucleoplasm. Identification of 348 human AluACA RNAs demonstrates that intron-encoded AluACA RNAs represent a novel, large subgroup of H/ACA RNAs, which are apparently confined to human or primate cells.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Human box H/ACA and Alu RNAs. (A) Structure and function of human box H/ACA pseudouridylation guide RNAs. The highly and moderately conserved nucleotides in the H, ACA, and CAB box motifs are in capital and lowercase letters, respectively. (N) Any nucleotide; (Ψ) uridines selected for pseudouridylation in the substrate RNA. The distance between the selected uridine and the H or ACA box of the guide RNA is ∼14 nt. (B) Organization and expression of Pol III-specific Alu RNA genes. The upstream enhancer, the internal A and B promoter, and the downstream terminator elements are indicated. The internal and terminal A-rich regions (An) are shown. Schematic structures of the full-length and 3′-terminally processed (scAlu) Alu RNAs are shown. The internal A-rich (A) and L regions required for Pol II inhibition are in shaded boxes.
Figure 2.
Figure 2.
Human AluACA RNAs. (A) Organization of human intronic AluACA RNA genes. Exons (Ex and Ex+1) of the host gene and the A-rich sequences (An) following the left and right arms of the intronic full-length Alu element are indicated. Frequencies of the four ribonucleotides in the predicted H, pCAB, dCAB, and ACA motifs of 348 AluACA RNAs are indicated by the heights of the corresponding letters. The 5′-terminal hairpin regions of AluACA RNAs are not included. (B) Proposed two-dimensional structures of the 3′ end regions of human AluACA RNAs. For each Alu subgroup, one sample RNA represented by the highest number of sequence reads is shown. The conserved H and ACA box motifs are in red, and the proximal and distal CAB boxes (pCAB and dCAB) are highlighted in blue. Potential pseudouridylation guide sequences are in green. Nucleotides located 14 nt upstream of the ACA box are indicated. Numbering of AluACA7, AluACA15, AluACA17, AluACA21, and AluACA177 RNAs was based on mappings of transiently expressed RNAs (see Fig. 4).
Figure 3.
Figure 3.
Processing of AluACA7 RNA from the second intron of transiently expressed β-globin pre-mRNA. (A) Schematic structure of the pGL/ALU7 expression vector. The cytomegalovirus promoter (CMV), the exon regions of the β-globin gene (E1–E3), the polyadenylation site (PA), and the SP6 promoter used for synthesis of antisense RNA probes are indicated. The open arrow indicates the full-length dimeric ALUACA7 DNA, inserted into the second intron of the human β-globin gene. The broken arrow represents the 3′-terminal right arm of the ALUACA7 Alu element. Relevant restriction sites are shown. (H) HindIII; (C) ClaI; (X) XhoI. The antisense RNA probes (probes 1–4), together with the lengths of globin exons, are shown. Nucleotide alterations introduced into the wild-type (WT) box H, pCAB, dCAB, and ACA sequence motifs of ALUACA7 are indicated. (B) In vivo processing of AluACA7 from the globin pre-mRNA. Cellular RNAs extracted from HeLa cells nontranfected (NT) or transfected with the pGL, pGL/ALU7, or pGL/ALU73′ expression plasmids were mapped by RNase protection performed with antisense RNAs indicated above the lanes. Probe RNAs protected by the overexpressed AluACA7 RNA and the processed exons (E1–E3) of the globin mRNA were separated on a 6% sequencing gel and visualized by autoradiography. (Lane M) Size markers in nucleotides. (C) Expression of AluACA7 depends on the H and ACA boxes. RNAs from HeLa cells transfected with the indicated expression plasmids were mapped with sequence-specific RNA probes. (D) Detection of HeLa endogenous AluACA7 RNA. HeLa cellular RNA (30 μg) was mapped with a high-specific-activity RNA probe complementary to the ALUACA7 DNA. Probe RNA fragments protected by AluACA7 or partially protected by other AluACA and Pol III-transcribed Alu RNAs were separated on a sequencing gel. Control mapping with Escherichia coli tRNA is shown.
Figure 4.
Figure 4.
Mapping of transiently overexpressed AluACA RNAs. (A) Determination of the 5′ terminus of transiently overexpressed AluACA7 with primer extension analysis. The 5′-terminal nucleotides of AluACA7 are indicated. (B) Mapping of the 3′ terminus of AluACA7 with RNA 3′ end race. The 3′-terminal AluACA7 and the 5′-terminal oligonucleotide tag sequences are shown. (C) Proposed secondary structures of the 5′ hairpins of AluACA RNAs. The 5′-terminal sequences of AluACA15, AluACA17, AluACA21, and AluACA117 were inferred from the lengths of transiently expressed RNAs measured by RNase mappings. The predicted H boxes are indicated. Nucleotides derived from left Alu arms or from upstream non-Alu-flanking sequences are shown. (D) Transient expression of AluACA RNAs in HeLa cells. RNAs from HeLa cells nontransfected (NT) or transfected with the indicated expression plasmids were analyzed by RNase mappings with sequence-specific probes indicated above the lanes. For other details, see the legend for Figure 3B.
Figure 5.
Figure 5.
Protein composition of AluACA RNPs. (A) Association of HeLa endogenous AluACA7 and AluACA15 RNAs with H/ACA core proteins and Wdr79. Extracts prepared from HeLa cells nontransfected (NT) or transfected with pFL-DYS, pNHP2-GFP, pGAR1-GFP, or pNOP10-GFP were subjected to IP with the indicated antibodies. IP of transiently expressed FL-Dys, Nhp2-GFP, Gar1-GFP, and Nop10-GFP proteins and endogenous HeLa Wdr79 and Srp14 was monitored by Western blot analysis. RNAs prepared from cell extracts (Ext) and the pellets of IP reactions were analyzed by RNase mapping with sequence-specific RNA probes indicated on the right. (Lane M) DNA and protein size markers. (B) Association of transiently overexpressed AluACA RNAs with scaRNP proteins. HeLa cells transfected with pGL/ALU7 or pGL/ALU15 were cotransfected with the pFL-DYS or pFL-WDR expression plasmids as indicated. Extracts prepared from nontransfected (NT) or transfected cells were subjected to IP with the indicated antibodies. RNAs prepared from cell extracts and pellets of IP reactions were analyzed by RNase protection.
Figure 6.
Figure 6.
The CAB boxes of AluACA RNAs function in Wdr79 binding. (A) The proximal and distal CAB boxes of AluACA7 bind Wdr79 in a cumulative manner. Nucleotide alterations introduced into the proximal and distal CAB box of AluACA7 are indicated. Extracts prepared from HeLa cells transfected with the pGL/ALU7 (WT), pGL/ALU7pCAB, pGL/ALU7dCAB, or pGL/ALU7pdCAB expression plasmids were reacted with an anti-Wdr79 antibody. RNAs isolated from cell extracts and the pellets of IP reactions were analyzed by RNase mappings. (B) The CAB boxes of AluACA RNAs are essential for both Wdr79 binding and efficient RNA accumulation. Schematic structure of the U64-AluACA7 composite RNA is shown. Extracts prepared from HeLa cells nontransfected (NT) or transfected with pGL/U64-ALU7 (WT) or pGL/U64-ALU7pdCAB were reacted with an anti-Wdr79 antibody. RNAs isolated from cell extracts and the pellets of IP reactions were analyzed by RNase protection. The first exon (E1) of globin mRNA is indicated.
Figure 7.
Figure 7.
Subcellular localization of AluACA RNAs. (A) HeLa AluACA7 and AluACA15 RNAs accumulate in the nucleus. RNA samples isolated from either HeLa cells (T) or the nuclear (Nu) and cytoplasmic (Cy) fractions of HeLa cells were mapped by RNase A/T1 protection with antisense RNA probes as indicated on the right. (Lane C) Control mapping with E. coli tRNA. (B) Expression of internally tagged AluACA7 RNAs in HeLa cells. Schematic structures of transiently expressed AluACA7-MSh and AluACA7-MS5′ RNAs with the inserted MS2 coat protein-binding motifs (in red) are shown. For the structure of U64-AluACA7, see Figure 6B. Extracts (Ext) prepared from HeLa cells nontransfected (NT) or transfected (TR) with the indicated combination of the pGL/ALU7-MSh, pGL/ALU7-MS5′ pGL/U64-ALU7, pFL-DYS, and pFLWDR79 expression plasmids were reacted with an anti-Flag antibody. RNAs prepared from cell extracts or the pellets of IP reactions were analyzed by RNase mapping. Lane Cont shows mapping with E. coli tRNA. (C) Fluorescent in situ hybridization. HeLa cells transiently expressing the indicated tagged or chimeric version of AluACA7 RNA were stained with sequence-specific fluorescent oligonucleotide probes. Nucleoli were visualized by expression of fibrillarin-GFP or Gar1-GFP. CBs were immunostained with antibodies against Wdr79 or coilin. DAPI staining of nuclear DNA was omitted at U2 localization. Bar, 10 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Atzorn V, Fragapane P, Kiss T 2004. U17/snR30 is a ubiquitous snoRNA with two conserved sequence motifs essential for 18S rRNA production. Mol Cell Biol 24: 1769–1778 - PMC - PubMed
    1. Baker DL, Youssef OA, Chastkofsky MI, Dy DA, Terns RM, Terns MP 2005. RNA-guided RNA modification: Functional organization of the archaeal H/ACA RNP. Genes Dev 19: 1238–1248 - PMC - PubMed
    1. Balakin AG, Smith L, Fournier MJ 1996. The RNA world of the nucleolus: Two major families of small RNAs defined by different box elements with related functions. Cell 86: 823–834 - PubMed
    1. Batzer MA, Deininger PL 2002. Alu repeats and human genomic diversity. Nat Rev Genet 3: 370–379 - PubMed
    1. Berger A, Strub K 2011. Multiple roles of Alu-related noncoding RNAs. Prog Mol Subcell Biol 51: 119–146 - PubMed

Publication types

LinkOut - more resources