Localization and structure of endonuclease cleavage sites involved in the processing of the rat 32S precursor to ribosomal RNA

The initial endonuclease cleavage site in 32 S pre-rRNA (precursor to rRNA) is located within the rate rDNA sequence by S1-nuclease protection mapping of purified nucleolar 28 S rRNA and 12 S pre-rRNA. The heterogeneous 5′- and 3′-termini of these rRNA abut and map within two CTC motifs in tSi2 (internal transcribed spacer 2) located at 50-65 and 4-20 base-pairs upstream from the homogeneous 5′-end of the 28 S rRNA gene. These results show that multiple endonuclease cleavages occur at CUC sites in tSi2 to generate 28 S rRNA and 12 S pre-rRNA with heterogeneous 5′- and 3′-termini, respectively. These molecules have to be processed further to yield mature 28 S and 5.8 S rRNA. Thermal-denaturation studies revealed that the base-pairing association in the 12 S pre-rRNA:28 S rRNA complex is markedly stronger than that in the 5.8 S:28 S rRNA complex. The sequence of about one-quarter (1322 base-pairs) of the 5′-part of the rat 28 S rDNA was determined. A computer search reveals the possibility that the cleavage sites in the CUC motifs are single-stranded, flanked by strongly base-paired GC tracts, involving tSi2 and 28 S rRNA sequences. The subsequent nuclease cleavages, generating the termini of mature rRNA, seem to be directed by secondary-structure interactions between 5.8 S and 28 S rRNA segments in pre-rRNA. An analysis for base-pairing among evolutionarily conserved sequences in 32 S pre-rRNA suggests that the cleavages yielding mature 5.8 S and 28 S rRNA are directed by base-pairing between (i) the 3′-terminus of 5.8 S rRNA and the 5′-terminus of 28 S rRNA and (ii) the 5′-terminus of 5.8 S rRNA and internal sequences in domain I of 28 S rRNA. A general model for primary- and secondary-structure interactions in pre-rRNA processing is proposed, and its implications for ribosome biogenesis in eukaryotes are briefly discussed.

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An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609550113 ◽

2016 ◽

Vol 113 (41) ◽

pp. E6126-E6134 ◽

Cited By ~ 19

Author(s):

Sunil Shetty ◽

Umesh Varshney

Keyword(s):

Escherichia Coli ◽

16S Rrna ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Complex Function ◽

Cold Sensitivity ◽

Base Pairs ◽

Initiation Complex ◽

Evolutionarily Conserved ◽

Multistep Process

Ribosome biogenesis, a complex multistep process, results in correct folding of rRNAs, incorporation of >50 ribosomal proteins, and their maturation. Deficiencies in ribosome biogenesis may result in varied faults in translation of mRNAs causing cellular toxicities and ribosomopathies in higher organisms. How cells ensure quality control in ribosome biogenesis for the fidelity of its complex function remains unclear. Using Escherichia coli, we show that initiator tRNA (i-tRNA), specifically the evolutionarily conserved three consecutive GC base pairs in its anticodon stem, play a crucial role in ribosome maturation. Deficiencies in cellular contents of i-tRNA confer cold sensitivity and result in accumulation of ribosomes with immature 3′ and 5′ ends of the 16S rRNA. Overexpression of i-tRNA in various strains rescues biogenesis defects. Participation of i-tRNA in the first round of initiation complex formation licenses the final steps of ribosome maturation by signaling RNases to trim the terminal extensions of immature 16S rRNA.

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Improved RNA secondary structure and tertiary base-pairing prediction using evolutionary profile, mutational coupling and two-dimensional transfer learning

Bioinformatics ◽

10.1093/bioinformatics/btab165 ◽

2021 ◽

Author(s):

Jaswinder Singh ◽

Kuldip Paliwal ◽

Tongchuan Zhang ◽

Jaspreet Singh ◽

Thomas Litfin ◽

...

Keyword(s):

High Resolution ◽

Secondary Structure ◽

Transfer Learning ◽

Rna Secondary Structure ◽

Computational Prediction ◽

Supplementary Information ◽

Base Pairing ◽

Base Pairs ◽

Homologous Sequences ◽

Non Coding Rnas

Abstract Motivation The recent discovery of numerous non-coding RNAs (long non-coding RNAs, in particular) has transformed our perception about the roles of RNAs in living organisms. Our ability to understand them, however, is hampered by our inability to solve their secondary and tertiary structures in high resolution efficiently by existing experimental techniques. Computational prediction of RNA secondary structure, on the other hand, has received much-needed improvement, recently, through deep learning of a large approximate data, followed by transfer learning with gold-standard base-pairing structures from high-resolution 3-D structures. Here, we expand this single-sequence-based learning to the use of evolutionary profiles and mutational coupling. Results The new method allows large improvement not only in canonical base-pairs (RNA secondary structures) but more so in base-pairing associated with tertiary interactions such as pseudoknots, non-canonical and lone base-pairs. In particular, it is highly accurate for those RNAs of more than 1000 homologous sequences by achieving >0.8 F1-score (harmonic mean of sensitivity and precision) for 14/16 RNAs tested. The method can also significantly improve base-pairing prediction by incorporating artificial but functional homologous sequences generated from deep mutational scanning without any modification. The fully automatic method (publicly available as server and standalone software) should provide the scientific community a new powerful tool to capture not only the secondary structure but also tertiary base-pairing information for building three-dimensional models. It also highlights the future of accurately solving the base-pairing structure by using a large number of natural and/or artificial homologous sequences. Availability and implementation Standalone-version of SPOT-RNA2 is available at https://github.com/jaswindersingh2/SPOT-RNA2. Direct prediction can also be made at https://sparks-lab.org/server/spot-rna2/. The datasets used in this research can also be downloaded from the GITHUB and the webserver mentioned above. Supplementary information Supplementary data are available at Bioinformatics online.

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Complex RNA Secondary Structures Mediate Mutually Exclusive Splicing of Coleoptera Dscam1

Frontiers in Genetics ◽

10.3389/fgene.2021.644238 ◽

2021 ◽

Vol 12 ◽

Author(s):

Haiyang Dong ◽

Lei Li ◽

Xiaohua Zhu ◽

Jilong Shi ◽

Ying Fu ◽

...

Keyword(s):

Secondary Structure ◽

Structure Prediction ◽

Secondary Structure Prediction ◽

Protein Isoforms ◽

Base Pairing ◽

Variable Exon ◽

Evolutionarily Conserved ◽

Exon 9 ◽

Exon 4 ◽

Docking Sites

Mutually exclusive splicing is an important mechanism for expanding protein diversity. An extreme example is the Down syndrome cell adhesion molecular (Dscam1) gene of insects, containing four clusters of variable exons (exons 4, 6, 9, and 17), which potentially generates tens of thousands of protein isoforms through mutually exclusive splicing, of which regulatory mechanisms are still elusive. Here, we systematically analyzed the variable exon 4, 6, and 9 clusters of Dscam1 in Coleoptera species. Through comparative genomics and RNA secondary structure prediction, we found apparent evidence that the evolutionarily conserved RNA base pairing mediates mutually exclusive splicing in the Dscam1 exon 4 cluster. In contrast to the fly exon 6, most exon 6 selector sequences in Coleoptera species are partially located in the variable exon region. Besides, bidirectional RNA–RNA interactions are predicted to regulate the mutually exclusive splicing of variable exon 9 of Dscam1. Although the docking sites in exon 4 and 9 clusters are clade specific, the docking sites-selector base pairing is conserved in secondary structure level. In short, our result provided a mechanistic framework for the application of long-range RNA base pairings in regulating the mutually exclusive splicing of Coleoptera Dscam1.

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Base pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar Ribonucleoproteins

10.1101/569061 ◽

2019 ◽

Author(s):

Erin Katelyn Kelly ◽

Dominic Philip Czekay ◽

Ute Kothe

Keyword(s):

Messenger Rna ◽

Ribosome Biogenesis ◽

Small Nuclear Rna ◽

Base Pairing ◽

Base Pairs ◽

Guide Rna ◽

Guide Rnas ◽

Pairing Strength ◽

Pairing Interactions ◽

Time Courses

AbstractH/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing interactions between H/ACA guide and substrate RNA. Besides ribosomal RNA (rRNA) and small nuclear RNA (snRNA), H/ACA snoRNPs are thought to also modify messenger RNA (mRNA) with potential impacts on gene expression. However, the base-pairing between known target RNAs and H/ACA guide RNAs varies widely in nature, and therefore the rules governing substrate RNA selection are still not fully understood. To provide quantitative insight into substrate RNA recognition, we systematically altered the sequence of a substrate RNA target by the Saccharomyces cerevisiae H/ACA guide RNA snR34. Time courses measuring pseudouridine formation revealed a gradual decrease in the initial velocity of pseudouridylation upon reducing the number of base pairs between substrate and guide RNA. Changing or inserting nucleotides close to the target uridine severely impairs pseudouridine formation. Interestingly, filter binding experiments show that all substrate RNA variants bind to H/ACA snoRNPs with nanomolar affinity. Next, we showed that binding of inactive, near-cognate RNAs to H/ACA snoRNPs does not inhibit their activity for cognate RNAs, presumably because near-cognate RNAs dissociate rapidly. We discuss that the modulation of initial velocities by the base pairing strength might affect the order and efficiency of pseudouridylation in rRNA during ribosome biogenesis. Moreover, the binding of H/ACA snoRNPs to near-cognate RNAs may be a mechanism to search for cognate target sites. Together, our data provide critical information to aid in the prediction of productive H/ACA guide – substrate RNA pairs.

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Two distant and precisely positioned domains promote transcription of Xenopus laevis rRNA genes: analysis with linker-scanning mutants

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4585-4593.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4585-4593

Author(s):

J J Windle ◽

B Sollner-Webb

Keyword(s):

Xenopus Laevis ◽

Promoter Activity ◽

Promoter Sequence ◽

Rrna Genes ◽

Rrna Gene ◽

Unexpected Result ◽

Base Pairs ◽

S1 Nuclease ◽

Transcriptional Initiation ◽

Upstream Promoter

To examine the internal organization of the promoter of the Xenopus laevis rRNA gene, we constructed a series of linker-scanning mutants that traverse the rDNA initiation region. The mutant genes, which have 3 to 11 clustered base substitutions set within an otherwise unaltered rDNA promoter sequence, were injected into Xenopus oocyte nuclei, and their transcriptional capacity was assessed by S1 nuclease analysis of the resultant RNA. The data demonstrate that there are two essential promoter domains, the distal boundaries of which coincide with the promoter boundaries established previously by analysis of 5' and 3' deletion mutants. The upstream promoter domain is relatively small and extends from residues ca. -140 to -128. The downstream domain is considerably larger, encompassing residues ca. -36 to +10, and exactly corresponds in both size and position to the mammalian minimal promoter region. The Xenopus rDNA sequence between these two essential domains has a much smaller effect on the level of transcriptional initiation. In light of the fact that a large portion of this intervening region consists of a segment (residues -114 to -72) that is duplicated many times in the upstream spacer to form an rDNA enhancer sequence, it is noteworthy that a "-115/-77 linker scanner," in which virtually this entire segment is replaced by a polylinker sequence, has full promoter activity in the injected Xenopus borealis oocytes. Analysis of a parallel series of spacing change linker-scanning mutants revealed the unexpected result that the relative positions of the upstream and downstream promoter domains are very critical: all spacing alterations of more than 2 base pairs within this 100-base-pair region virtually abolish promoter activity. We conclude that the factors that bind to these two distant promoter domains must interact in a very precise stereospecific manner.

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Two distant and precisely positioned domains promote transcription of Xenopus laevis rRNA genes: analysis with linker-scanning mutants.

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4585 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4585-4593 ◽

Cited By ~ 17

Author(s):

J J Windle ◽

B Sollner-Webb

Keyword(s):

Xenopus Laevis ◽

Promoter Activity ◽

Promoter Sequence ◽

Rrna Genes ◽

Rrna Gene ◽

Unexpected Result ◽

Base Pairs ◽

S1 Nuclease ◽

Transcriptional Initiation ◽

Upstream Promoter

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Base pairing structure in the poly d(G-T) double helix: wobble base pairs

Nucleic Acids Research ◽

10.1093/nar/5.6.1955 ◽

1978 ◽

Vol 5 (6) ◽

pp. 1955-1970 ◽

Cited By ~ 41

Author(s):

Thomas A. Early ◽

John Olmsted ◽

David R. Kearns ◽

Axel G. Lezius

Keyword(s):

Double Helix ◽

Base Pairing ◽

Base Pairs

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Location of the initial cleavage sites in mouse pre-rRNA.

Molecular and Cellular Biology ◽

10.1128/mcb.3.8.1501 ◽

1983 ◽

Vol 3 (8) ◽

pp. 1501-1510 ◽

Cited By ~ 38

Author(s):

L H Bowman ◽

W E Goldman ◽

G I Goldberg ◽

M B Hebert ◽

D Schlessinger

Keyword(s):

Blot Hybridization ◽

Northern Blot Hybridization ◽

28S Rrna ◽

Cleavage Sites ◽

S1 Nuclease ◽

5.8S Rrna ◽

Initial Cleavage ◽

S1 Nuclease Mapping ◽

Mapping Techniques ◽

Nuclease Mapping

The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques. These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA. Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream. This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini. Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA. Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms. Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W. E. Goldman, G. Goldberg, L. H. Bowman, D. Steinmetz, and D. Schlessinger, Mol. Cell. Biol. 3:1488-1500, 1983).

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DNA damage and heat shock dually regulate genes in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.90 ◽

1986 ◽

Vol 6 (1) ◽

pp. 90-96 ◽

Cited By ~ 39

Author(s):

T McClanahan ◽

K McEntee

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Heat Shock ◽

Shock Treatment ◽

Northern Hybridization ◽

Heat Shock Treatment ◽

Base Pairs ◽

S1 Nuclease ◽

Hsp70 Family ◽

Transcriptional Start Sites

Two Saccharomyces cerevisiae genes isolated in a differential hybridization screening for DNA damage regulation (DDR genes) were also transcriptionally regulated by heat shock treatment. A 0.45-kilobase transcript homologous to the DDRA2 gene and a 1.25-kilobase transcript homologous to the DDR48 gene accumulated after exposure of cells to 4-nitroquinoline-1-oxide (NQO; 1 to 1.5 microgram/ml) or brief heat shock (20 min at 37 degrees C). The DDRA2 transcript, which was undetectable in untreated cells, was induced to high levels by these treatments, and the DDR48 transcript increased more than 10-fold as demonstrated by Northern hybridization analysis. Two findings argue that dual regulation of stress-responsive genes is not common in S. cerevisiae. First, two members of the heat shock-inducible hsp70 family of S. cerevisiae, YG100 and YG102, were not induced by exposure to NQO. Second, at least one other DNA-damage-inducible gene, DIN1, was not regulated by heat shock treatment. We examined the structure of the induced RNA homologous to DDRA2 after heat shock and NQO treatments by S1 nuclease protection experiments. Our results demonstrated that the DDRA2 transcript initiates equally frequently at two sites separated by 5 base pairs. Both transcriptional start sites were utilized when cells were exposed to either NQO or heat shock treatment. These results indicate that DDRA2 and DDR48 are members of a unique dually regulated stress-responsive family of genes in S. cerevisiae.

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