Systematic Study of Sequence Motifs for RNA trans Splicing in Trypanosoma brucei

ABSTRACT mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5′ splice sites, branch points, pyrimidine-rich regions [poly(Y) tracts], 3′ splice sites (3′SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in the poly(Y) tract and around the 3′SS, we constructed a luciferase-β-galactosidase double-reporter system. By testing ∼90 sequences, we demonstrated that the optimum poly(Y) tract length is ∼25 nucleotides. Interspersing a purely uridine-containing poly(Y) tract with cytidine resulted in increased trans-splicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3′SS is important, and an AC dinucleotide at positions −3 and −4 can lead to a 20-fold decrease in trans splicing. However, efficient trans splicing can be restored by inserting a second AG dinucleotide downstream, which does not function as a splice site but may aid in recruitment of the splicing machinery. These findings should assist in the development of improved algorithms for computationally identifying a 3′SS and help to discriminate noncoding open reading frames from true genes in current efforts to annotate the T. brucei genome.

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Stress-Inducible Alternative Translation Initiation of Human Cytomegalovirus Latency Protein pUL138

Journal of Virology ◽

10.1128/jvi.00855-10 ◽

2010 ◽

Vol 84 (18) ◽

pp. 9472-9486 ◽

Cited By ~ 48

Author(s):

Lora Grainger ◽

Louis Cicchini ◽

Michael Rak ◽

Alex Petrucelli ◽

Kerry D. Fitzgerald ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Translation Initiation ◽

Rna Splicing ◽

Mammalian Cells ◽

Open Reading Frames ◽

Reporter System ◽

Entry Site ◽

Sequence Element ◽

Low Passage

ABSTRACT We have previously characterized a 21-kDa protein encoded by UL138 (pUL138) as a viral factor inherent to low-passage strains of human cytomegalovirus (HCMV) that is required for latent infection in vitro. pUL138 is encoded on 3.6-, 2.7-, and 1.4-kb 3′ coterminal transcripts that are produced during productive and latent infections. pUL138 is encoded at the 3′ end of each transcript and is preceded by an extensive 5′ sequence (∼0.5 to 2.5 kb) containing several putative open reading frames (ORFs). We determined that three putative ORFs upstream of UL138 (UL133, UL135, and UL136) encode proteins. The UL138 transcripts are polycistronic, such that each transcript expresses pUL138 in addition to the most-5′ ORF. The upstream coding sequences (CDS) present a significant challenge for the translation of pUL138 in mammalian cells. We hypothesized that sequences 5′ of UL138 mediate translation initiation of pUL138 by alternative strategies. Accordingly, a 663-nucloetide (nt) sequence overlapping the UL136 CDS supported expression of a downstream cistron in a bicistronic reporter system. We did not detect cryptic promoter activity or RNA splicing events that could account for downstream cistron expression. These data are consistent with the sequence element functioning as an internal ribosome entry site (IRES). Interestingly, pUL138 expression from the 3.6- and 2.7-kb transcripts was induced by serum stress, which concomitantly inhibited normal cap-dependent translation. Our work suggests that an alternative and stress-inducible strategy of translation initiation ensures expression of pUL138 under a variety of cellular contexts. The UL138 polycistronic transcripts serve to coordinate the expression of multiple proteins, including a viral determinant of HCMV latency.

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Splicing at the phase-separated nuclear speckle interface: a model

Nucleic Acids Research ◽

10.1093/nar/gkaa1209 ◽

2020 ◽

Author(s):

Susan E Liao ◽

Oded Regev

Keyword(s):

Phase Separation ◽

Rna Splicing ◽

Splicing Factors ◽

Sequence Motifs ◽

Splice Sites ◽

Nuclear Speckles ◽

Nuclear Speckle ◽

Functional Roles ◽

Hnrnp Proteins ◽

Model Sequence

Abstract Phase-separated membraneless bodies play important roles in nucleic acid biology. While current models for the roles of phase separation largely focus on the compartmentalization of constituent proteins, we reason that other properties of phase separation may play functional roles. Specifically, we propose that interfaces of phase-separated membraneless bodies could have functional roles in spatially organizing biochemical reactions. Here we propose such a model for the nuclear speckle, a membraneless body implicated in RNA splicing. In our model, sequence-dependent RNA positioning along the nuclear speckle interface coordinates RNA splicing. Our model asserts that exons are preferentially sequestered into nuclear speckles through binding by SR proteins, while introns are excluded through binding by nucleoplasmic hnRNP proteins. As a result, splice sites at exon-intron boundaries are preferentially positioned at nuclear speckle interfaces. This positioning exposes splice sites to interface-localized spliceosomes, enabling the subsequent splicing reaction. Our model provides a simple mechanism that seamlessly explains much of the complex logic of splicing. This logic includes experimental results such as the antagonistic duality between splicing factors, the position dependence of splicing sequence motifs, and the collective contribution of many motifs to splicing decisions. Similar functional roles for phase-separated interfaces may exist for other membraneless bodies.

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Sequence determinants and evolution of constitutive and alternative splicing in yeast species

10.1101/2020.04.20.050609 ◽

2020 ◽

Author(s):

Dvir Schirman ◽

Zohar Yakhini ◽

Orna Dahan ◽

Yitzhak Pilpel

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Splice Site ◽

Rna Splicing ◽

Yeast Species ◽

Combinatorial Design ◽

Splice Sites ◽

Eukaryotic Gene ◽

Splicing Efficiency ◽

Splicing Machinery

RNA splicing is a key process in eukaryotic gene expression. Most Intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Using a combinatorial design of synthetic introns we demonstrate how non-consensus splice site sequences affect splicing efficiency in each of the three splice sites. We then show that S. cerevisiae splicing machinery tends to select alternative 3' splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns' 3' ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organism's splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, exposing intronic features that control and facilitate alternative splicing. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome.

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Spliceosomal Proteomics in Trypanosoma brucei Reveal New RNA Splicing Factors

Eukaryotic Cell ◽

10.1128/ec.00075-09 ◽

2009 ◽

Vol 8 (7) ◽

pp. 990-1000 ◽

Cited By ~ 36

Author(s):

Daniela Luz Ambrósio ◽

Ju Huck Lee ◽

Aswini K. Panigrahi ◽

Tu Ngoc Nguyen ◽

Regina Maria Barretto Cicarelli ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Rna Splicing ◽

Bioinformatic Analysis ◽

Specific Protein ◽

Splicing Factors ◽

Small Nuclear Ribonucleoprotein ◽

Mrna Maturation ◽

Trypanosomatid Parasites ◽

Specific Proteins ◽

Intron Removal

ABSTRACT In trypanosomatid parasites, spliced leader (SL) trans splicing is an essential nuclear mRNA maturation step which caps mRNAs posttranscriptionally and, in conjunction with polyadenylation, resolves individual mRNAs from polycistronic precursors. While all trypanosomatid mRNAs are trans spliced, intron removal by cis splicing is extremely rare and predicted to occur in only four pre-mRNAs. trans- and cis-splicing reactions are carried out by the spliceosome, which consists of U-rich small nuclear ribonucleoprotein particles (U snRNPs) and of non-snRNP factors. Mammalian and yeast spliceosome complexes are well characterized and found to be associated with up to 170 proteins. Despite the central importance of trans splicing in trypanosomatid gene expression, only the core RNP proteins and a few snRNP-specific proteins are known. To characterize the trypanosome spliceosomal protein repertoire, we conducted a proteomic analysis by tagging and tandem affinity-purifying the canonical core RNP protein SmD1 in Trypanosoma brucei and by identifying copurified proteins by mass spectrometry. The set of 47 identified proteins harbored nearly all spliceosomal snRNP factors characterized in trypanosomes thus far and 21 proteins lacking a specific annotation. A bioinformatic analysis combined with protein pull-down assays and immunofluorescence microscopy identified 10 divergent orthologues of known splicing factors, including the missing U1-specific protein U1A. In addition, a novel U5-specific, and, as we show, an essential splicing factor was identified that shares a short, highly conserved N-terminal domain with the yeast protein Cwc21p and was thus tentatively named U5-Cwc21. Together, these data strongly indicate that most of the identified proteins are components of the spliceosome.

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Analysis of the Genetic Variability of Virulence-Related Loci in Epidemic Clones of Methicillin-Resistant Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.1.366-379.2005 ◽

2005 ◽

Vol 49 (1) ◽

pp. 366-379 ◽

Cited By ~ 41

Author(s):

A. R. Gomes ◽

S. Vinga ◽

M. Zavolan ◽

H. de Lencastre

Keyword(s):

Staphylococcus Aureus ◽

Genetic Variability ◽

Amino Acid Level ◽

Point Of View ◽

Sequence Motifs ◽

Related Factors ◽

Specific Sequence ◽

Methicillin Resistant ◽

Sequence Types ◽

R Domain

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) isolates have previously been classified into major epidemic clonal types by pulsed-field gel electrophoresis in combination with multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec typing. We aimed to investigate whether genetic variability in potentially polymorphic domains of virulence-related factors could provide another level of differentiation in a diverse collection of epidemic MRSA clones. The target regions of strains representative of epidemic clones and genetically related methicillin-susceptible S. aureus isolates from the 1960s that were sequenced included the R domains of clfA and clfB; the D, W, and M regions of fnbA and fnbB; and three regions in the agr operon. Sequence variation ranged from very conserved regions, such as those for RNAIII and the agr interpromoter region, to the highly polymorphic R regions of the clf genes. The sequences of the clf R domains could be grouped into six major sequence types on the basis of the sequences in their 3′ regions. Six sequence types were also observed for the fnb sequences at the amino acid level. From an evolutionary point of view, it was interesting that a small DNA stretch at the 3′ clf R-domain sequence and the fnb sequences agreed with the results of MLST for this set of strains. In particular, clfB R-domain sequences, which had a high discriminatory capacity and with which the types distinguished were congruent with those obtained by other molecular typing methods, have potential for use for the typing of S. aureus. Clone- and strain-specific sequence motifs in the clf and fnb genes may represent useful additions to a typing methodology with a DNA array.

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Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics

Nature Communications ◽

10.1038/s41467-021-24674-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Bertrand Beckert ◽

Elodie C. Leroy ◽

Shanmugapriya Sothiselvam ◽

Lars V. Bock ◽

Maxim S. Svetlov ◽

...

Keyword(s):

Antibiotic Resistance Genes ◽

Structural Basis ◽

Sequence Motifs ◽

Specific Sequence ◽

Bacterial Ribosome ◽

Translational Arrest ◽

Exit Tunnel ◽

Mechanistic Basis ◽

Dynamics Simulations ◽

Context Specific

AbstractMacrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.

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Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotides

Scientific Reports ◽

10.1038/s41598-021-94639-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kristin A. Ham ◽

Niall P. Keegan ◽

Craig S. McIntosh ◽

May T. Aung-Htut ◽

Khine Zaw ◽

...

Keyword(s):

Rna Splicing ◽

Antisense Oligonucleotides ◽

Live Cells ◽

Splice Sites ◽

Cryptic Splice Site ◽

Exon Definition ◽

Rna Targets ◽

Cryptic Splice Sites ◽

Six Genes ◽

Intended Effect

AbstractAntisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon’s excluded segment.

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CDKN1C mutation in Wiedemann-Beckwith syndrome patients reduces RNA splicing efficiency and identifies a splicing enhancer

American Journal of Medical Genetics ◽

10.1002/ajmg.a.30020 ◽

2004 ◽

Vol 127A (3) ◽

pp. 268-276 ◽

Cited By ~ 8

Author(s):

Jocelyne M. Lew ◽

Yan Ling Fei ◽

Kirk Aleck ◽

Benjamin J. Blencowe ◽

Rosanna Weksberg ◽

...

Keyword(s):

Rna Splicing ◽

Splicing Efficiency ◽

Splicing Enhancer

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Genetic Interactions WithCLF1Identify Additional Pre-mRNA Splicing Factors and a Link Between Activators of Yeast Vesicular Transport and Splicing

Genetics ◽

10.1093/genetics/164.3.895 ◽

2003 ◽

Vol 164 (3) ◽

pp. 895-907 ◽

Cited By ~ 3

Author(s):

Kevin Vincent ◽

Qiang Wang ◽

Steven Jay ◽

Kathryn Hobbs ◽

Brian C Rymond

Keyword(s):

Mrna Splicing ◽

Splicing Factors ◽

Sequence Information ◽

Specific Sequence ◽

Synthetic Lethal ◽

Growth Defects ◽

Mrna Maturation ◽

Assembly Factor ◽

Gtpase Activation ◽

Synthetic Growth

AbstractClf1 is a conserved spliceosome assembly factor composed predominately of TPR repeats. Here we show that the TPR elements are not functionally equivalent, with the amino terminus of Clf1 being especially sensitive to change. Deletion and add-back experiments reveal that the splicing defect associated with TPR removal results from the loss of TPR-specific sequence information. Twelve mutants were found that show synthetic growth defects when combined with an allele that lacks TPR2 (i.e., clf1Δ2). The identified genes encode the Mud2, Ntc20, Prp16, Prp17, Prp19, Prp22, and Syf2 splicing factors and four proteins without established contribution to splicing (Bud13, Cet1, Cwc2, and Rds3). Each synthetic lethal with clf1Δ2 (slc) mutant is splicing defective in a wild-type CLF1 background. In addition to the splicing factors, SSD1, BTS1, and BET4 were identified as dosage suppressors of clf1Δ2 or selected slc mutants. These results support Clf1 function through multiple stages of the spliceosome cycle, identify additional genes that promote cellular mRNA maturation, and reveal a link between Rab/Ras GTPase activation and the process of pre-mRNA splicing.

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Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites

Molecular and Cellular Biology ◽

10.1128/mcb.4.5.966-972.1984 ◽

1984 ◽

Vol 4 (5) ◽

pp. 966-972

Author(s):

C Montell ◽

E F Fisher ◽

M H Caruthers ◽

A J Berk

Keyword(s):

Splice Site ◽

Rna Splicing ◽

Early Phase ◽

Late Phase ◽

Productive Infection ◽

Splice Sites ◽

Mrna Synthesis ◽

Adenovirus E1b ◽

Cryptic Splice Sites ◽

Rna Splice Sites

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

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