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
. 2009 Dec 1;106(48):20246-51.
doi: 10.1073/pnas.0911658106. Epub 2009 Nov 12.

Processing of multiple-intron-containing pretRNA

Giuseppe D Tocchini-Valentini  1 , Paolo Fruscoloni, Glauco P Tocchini-Valentini
Affiliations

Processing of multiple-intron-containing pretRNA

Giuseppe D Tocchini-Valentini et al. Proc Natl Acad Sci U S A. .

Abstract

Computational studies predict the simultaneous presence of two and even three introns in certain crenarchaeal tRNA genes. In these multiple-intron-containing pretRNAs, the introns are nested one inside the other and the pretRNA folds into a conformation that is anticipated to allow splicing of the last intron only after splicing the others. A set of operations, each consisting of two cleavages and one ligation, therefore needs to be carried out sequentially. PretRNAs containing multiple introns are predicted to fold, forming bulge-helix-bulge (BHB) and BHB-like motifs. The tRNA splicing endonuclease should recognize these motifs. To test this hypothetical scenario, we used the homotetrameric enzyme from Methanocaldococcus jannaschii (METJA) and the heterotetrameric enzyme from Sulfolobus solfataricus (SULSO). On the basis of our previous studies, the METJA enzyme should cleave only the BHB structure motif, while the SULSO enzyme can in addition cleave variant substrate structures, like the bulge-helix-loop (BHL). We show here that the processing of multiple-intron-containing pretRNA can be observed in vitro.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Processing of METTH pretRNAProII. (A) Predicted secondary structure with arrows indicating the splicing sites. The sequences of the two introns are represented in lowercase. (B) tRNA endonuclease assay. The reaction products are indicated. Lane 1, control (no enzyme); lane 2, homotetrameric METJA endonuclease; lane 3, heterotetrameric SULSO endonuclease.
Fig. 2.
Fig. 2.
Processing of METTH PretRNAProI. (A) Predicted secondary structure following first intron removal (pretRNAProII) and religation, with arrows indicating the splicing sites. The structure on the right corresponds to the actual structure determined by RNase T1 protection and tRNA endonuclease assay. (B) RNase protection assay. Lane1, control (no enzyme); lane 2, alkaline hydrolysis; lane 3, RNase T1 digest in denaturing conditions; lane 4, RNase T1 digest under native conditions. (C) tRNA endonuclease assay. The reaction products are indicated. Lane 1, control (no enzyme); lane 2, homotetrameric METJA endonuclease; lane 3, heterotetrameric SULSO endonuclease.
Fig. 3.
Fig. 3.
Processing of THERPE pretRNAProIII. (A) Predicted secondary structure; arrows are indicating the splicing sites, numbered starting from the 5′ phosphate of the transcript. (B and C) In the order, long and a short run electrophoresis of the same tRNA endonuclease assays performed on both 5′ labeled transcript (γ) or uniformly labeled trascript (α). The bands corresponding to each splice site are numbered, as previously specified. Lane 1, 2 control (no enzyme); lane 3, 4 homotetrameric METJA endonuclease; lane 5,6 RNase T1 digest and partial hydrolysis of the 5′ labeled transcript; lane 7,8 heterotetrameric SULSO endonuclease.
Fig. 4.
Fig. 4.
Processing of THERPE PretRNAProI. (A) Predicted secondary structure following the removal of two introns (pretRNAProIII) and religation, arrows are indicating the splicing sites. The structure on the right corresponds to the actual structure determined by tRNA endonuclease assay. (B) tRNA endonuclease assay. The reaction products are indicated. Lane 1, control (no enzyme); lane 2, homotetrameric METJA endonuclease; lane 3, heterotetrameric SULSO endonuclease.
Fig. 5.
Fig. 5.
Linear representation of the multiple-intron-containing pretRNA. The arrows indicate the splicing and ligation reactions.
Fig. 6.
Fig. 6.
Processing of HALMA pretRNAIleu/Trp. (A) Predicted secondary structure according to (19). The arrows indicate the predicted sites to obtain tRNAIle and tRNATrp, asterisks indicate the actual splicing sites. (B) tRNA endonuclease assay. The reaction products are indicated. Lane 1, control (no enzyme); lane 2, homotetrameric METJA endonuclease; lane 3, heterotetrameric SULSO endonuclease.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abelson J, Trotta CR, Li H. tRNA splicing. J Biol Chem. 1998;273:12685–12688. - PubMed
    1. Saldanha R, Mohr G, Belfort M, Lambowitz AM. Group I and group II introns. FASEB J. 1993;7:15–24. - PubMed
    1. Marck C, Grosjean H. Identification of BHB splicing motifs in intron-containing tRNAs from 18 archaea: Evolutionary implications. RNA. 2003;9:1516–1531. - PMC - PubMed
    1. Fabbri S, et al. Conservation of substrate recognition mechanisms by tRNA splicing endonucleases. Science. 1998;280:284–286. - PubMed
    1. Di Nicola Negri E, et al. The eucaryal tRNA splicing endonuclease recognizes a tripartite set of RNA elements. Cell. 1997;89:859–866. - PubMed

Publication types

LinkOut - more resources