In this review I focus on the role of splicing in long non-coding RNA (lncRNA) life. First, I summarize differences between the splicing efficiency of protein-coding genes and lncRNAs and discuss why non-coding RNAs are spliced less efficiently. In the second half of the review, I speculate why splice sites are the most conserved sequences in lncRNAs and what additional roles could splicing play in lncRNA metabolism. I discuss the hypothesis that the splicing machinery can, besides its dominant role in intron removal and exon joining, protect cells from undesired transcripts.

• Klíčová slova
• SR proteins, large intervening non-coding RNA, snRNP, spliceosomes, splicing,
• MeSH
• RNA dlouhá nekódující * genetika   MeSH
• sestřih RNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• RNA dlouhá nekódující * MeSH

Pre-mRNA splicing represents an important regulatory layer of eukaryotic gene expression. In the simple budding yeast Saccharomyces cerevisiae, about one-third of all mRNA molecules undergo splicing, and splicing efficiency is tightly regulated, for example, during meiotic differentiation. S. cerevisiae features a streamlined, evolutionarily highly conserved splicing machinery and serves as a favourite model for studies of various aspects of splicing. RNA-seq represents a robust, versatile, and affordable technique for transcriptome interrogation, which can also be used to study splicing efficiency. However, convenient bioinformatics tools for the analysis of splicing efficiency from yeast RNA-seq data are lacking. We present a complete workflow for the calculation of genome-wide splicing efficiency in S. cerevisiae using strand-specific RNA-seq data. Our pipeline takes sequencing reads in the FASTQ format and provides splicing efficiency values for the 5' and 3' splice junctions of each intron. The pipeline is based on up-to-date open-source software tools and requires very limited input from the user. We provide all relevant scripts in a ready-to-use form. We demonstrate the functionality of the workflow using RNA-seq datasets from three spliceosome mutants. The workflow should prove useful for studies of yeast splicing mutants or of regulated splicing, for example, under specific growth conditions.

• MeSH
• databáze nukleových kyselin MeSH
• mutace genetika   MeSH
• prekurzory RNA genetika   MeSH
• průběh práce * MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvenční analýza RNA metody   MeSH
• sestřih RNA genetika   MeSH
• spliceozomy genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• prekurzory RNA MeSH

Acceptor splice site recognition (3' splice site: 3'ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3'ss, of which U2AF35 has a dual function: (i) It binds to the intron-exon border of some 3'ss and (ii) mediates enhancer-binding splicing activators' interactions with the spliceosome. Alternative mechanisms for 3'ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3'ss recognition where the intron-exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3'ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3'ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons' inclusion. Most probably, both factors stochastically bind the 3'ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3'ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants' effects on splicing.

• Klíčová slova
• SRSF1, U2AF35, acceptor splice site recognition, pre-mRNA splicing, splicing enhancer,
• MeSH
• alternativní sestřih genetika   MeSH
• exony genetika   MeSH
• HeLa buňky MeSH
• introny genetika   MeSH
• lidé MeSH
• místa sestřihu RNA genetika   fyziologie   MeSH
• proteiny vázající RNA metabolismus   MeSH
• regulační oblasti nukleových kyselin genetika   MeSH
• sekvence nukleotidů genetika   MeSH
• serin-arginin sestřihové faktory metabolismus   MeSH
• sestřih RNA fyziologie   MeSH
• sestřihové faktory metabolismus   fyziologie   MeSH
• sestřihový faktor U2AF metabolismus   MeSH
• spliceozomy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• místa sestřihu RNA MeSH
• proteiny vázající RNA MeSH
• serin-arginin sestřihové faktory MeSH
• sestřihové faktory MeSH
• sestřihový faktor U2AF MeSH
• SRSF1 protein, human MeSH Prohlížeč
• U2AF1 protein, human MeSH Prohlížeč

Trans-splicing is a process by which 5'- and 3'-ends of two pre-RNA molecules transcribed from different sites of the genome can be joined together to form a single RNA molecule. The spliced leader (SL) trans-splicing is mediated by the spliceosome and it allows the replacement of 5'-end of pre-mRNA by 5'(SL)-end of SL-RNA. This form of splicing has been observed in many phylogenetically unrelated eukaryotes. Either the SL trans-splicing (SLTS) originated in the last eukaryotic common ancestor (LECA) (or even earlier) and it was lost in most eukaryotic lineages, or this mechanism of RNA processing evolved several times independently in various unrelated eukaryotic taxa. The bioinformatic comparisons of SL-RNAs from various eukaryotic taxonomic groups have revealed the similarities of secondary structures of most SL-RNAs and a relative conservation of their splice sites (SSs) and Sm-binding sites (SmBSs). We propose that such structural and functional similarities of SL-RNAs are unlikely to have evolved repeatedly many times. Hence, we favor the scenario of an early evolutionary origin for the SLTS and multiple losses of SL-RNAs in various eukaryotic lineages.

• Klíčová slova
• Intron, RNA secondary structure, SL-RNA, Sm-binding site, Spliceosome,
• MeSH
• Eukaryota genetika   metabolismus   MeSH
• fylogeneze MeSH
• molekulární evoluce * MeSH
• prekurzory RNA metabolismus   MeSH
• RNA se sestřihovou vedoucí sekvencí genetika   metabolismus   MeSH
• trans-splicing * MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• prekurzory RNA MeSH
• RNA se sestřihovou vedoucí sekvencí MeSH

TDP-43 encodes an alternative-splicing regulator with tandem RNA-recognition motifs (RRMs). The protein regulates cystic fibrosis transmembrane regulator (CFTR) exon 9 splicing through binding to long UG-rich RNA sequences and is found in cytoplasmic inclusions of several neurodegenerative diseases. We solved the solution structure of the TDP-43 RRMs in complex with UG-rich RNA. Ten nucleotides are bound by both RRMs, and six are recognized sequence specifically. Among these, a central G interacts with both RRMs and stabilizes a new tandem RRM arrangement. Mutations that eliminate recognition of this key nucleotide or crucial inter-RRM interactions disrupt RNA binding and TDP-43-dependent splicing regulation. In contrast, point mutations that affect base-specific recognition in either RRM have weaker effects. Our findings reveal not only how TDP-43 recognizes UG repeats but also how RNA binding-dependent inter-RRM interactions are crucial for TDP-43 function.

• MeSH
• DNA vazebné proteiny chemie   metabolismus   fyziologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• protein CFTR genetika   metabolismus   MeSH
• proteiny vázající RNA chemie   metabolismus   fyziologie   MeSH
• sekvence aminokyselin MeSH
• sestřih RNA fyziologie   MeSH
• terciární struktura proteinů MeSH
• vazebná místa MeSH
• zastoupení bazí MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CFTR protein, human MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• protein CFTR MeSH
• proteiny vázající RNA MeSH

There are numerous data suggesting that two key steps in gene expression-transcription and splicing influence each other closely. For a long time it was known that chromatin modifications regulate transcription, but only recently it was shown that chromatin and histone modifications play a significant role in pre-mRNA splicing. Here we summarize interactions between splicing machinery and chromatin and discuss their potential functional significance. We focus mainly on histone acetylation and methylation and potential mechanisms of their role in splicing. It seems that whereas histone acetylation acts mainly by alterating the transcription rate, histone methylation can also influence splicing directly by recruiting various splicing components.

• Klíčová slova
• alternative splicing, chromatin, exon, histone acetylation, histone methylation, nucleosome, snRNP, transcription,
• MeSH
• chromatin genetika   metabolismus   MeSH
• genetická transkripce genetika   MeSH
• lidé MeSH
• nukleoproteiny metabolismus   MeSH
• prekurzory RNA genetika   MeSH
• sestřih RNA * MeSH
• transkripční faktory metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• chromatin MeSH
• nukleoproteiny MeSH
• prekurzory RNA MeSH
• transkripční faktory MeSH

The ability of a cell to properly express its genes depends on optimal transcription and splicing. RNA polymerase II (RNAPII) transcribes protein-coding genes and produces pre-mRNAs, which undergo, largely co-transcriptionally, intron excision by the spliceosome complex. Spliceosome activation is a major control step, leading to a catalytically active complex. Recent work has showed that cyclin-dependent kinase (CDK)11 regulates spliceosome activation via the phosphorylation of SF3B1, a core spliceosome component. Thus, CDK11 arises as a major coordinator of gene expression in metazoans due to its role in the rate-limiting step of pre-mRNA splicing. This review outlines the evolution of CDK11 and SF3B1 and their emerging roles in splicing regulation. It also discusses how CDK11 and its inhibition affect transcription and cell cycle progression.

• Klíčová slova
• C-terminal domain of RNA polymerase II, OTS964, ULM–UHM interaction, cell cycle progression, constitutive splicing, histone transcription,
• MeSH
• buněčný cyklus MeSH
• cyklin-dependentní kinasy * metabolismus   genetika   MeSH
• lidé MeSH
• regulace genové exprese * MeSH
• sestřih RNA * genetika   MeSH
• sestřihové faktory MeSH
• spliceozomy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• cyklin-dependentní kinasy * MeSH
• sestřihové faktory MeSH

The essential components of splicing are the splicing factors accumulated in nuclear speckles; thus, we studied how DNA damaging agents and A-type lamin depletion affect the properties of these regions, positive on the SC-35 protein. We observed that inhibitor of PARP (poly (ADP-ribose) polymerase), and more pronouncedly inhibitors of RNA polymerases, caused DNA damage and increased the SC35 protein level. Interestingly, nuclear blebs, induced by PARP inhibitor and observed in A-type lamin-depleted or senescent cells, were positive on both the SC-35 protein and another component of the spliceosome, SRRM2. In the interphase cell nuclei, SC-35 interacted with the phosphorylated form of RNAP II, which was A-type lamin-dependent. In mitotic cells, especially in telophase, the SC35 protein formed a well-visible ring in the cytoplasmic fraction and colocalized with β-catenin, associated with the plasma membrane. The antibody against the SRRM2 protein showed that nuclear speckles are already established in the cytoplasm of the late telophase and at the stage of early cytokinesis. In addition, we observed the occurrence of splicing factors in the nuclear blebs and micronuclei, which are also sites of both transcription and splicing. This conclusion supports the fact that splicing proceeds transcriptionally. According to our data, this process is A-type lamin-dependent. Lamin depletion also reduces the interaction between SC35 and β-catenin in mitotic cells.

• Klíčová slova
• PARP inhibitor, RNA pol II, SC-35, splicing,
• MeSH
• HeLa buňky MeSH
• laminy metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• PARP inhibitory terapeutické užití   MeSH
• poly(ADP-ribosa)polymerasa 1 MeSH
• RNA-polymerasa II metabolismus   MeSH
• sestřihové faktory metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• laminy MeSH
• PARP inhibitory MeSH
• PARP1 protein, human MeSH Prohlížeč
• poly(ADP-ribosa)polymerasa 1 MeSH
• RNA-polymerasa II MeSH
• sestřihové faktory MeSH

Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of variants of uncertain significance (VUSs) found via whole-genome and exome sequencing can be difficult for genes that have low expression in the blood due to insufficient read count coverage aligned to specific genes of interest. Here, we present a short amplicon reverse transcription-polymerase chain reaction(RT-PCR) for the detection of genes with low blood expression. Short amplicon RT-PCR, is designed to span three exons where an exon harboring a variant is flanked by one upstream and one downstream exon. We tested short amplicon RT-PCRs for genes that have median transcripts per million (TPM) values less than one according to the genotype-tissue expression database. Median TPM values of genes analyzed in this study are SYN1 = 0.8549, COL1A1 = 0.6275, TCF4 = 0.4009, DSP = .2894, TTN = 0.2851, COL5A2 = 0.1036, TERT = 0.04452, NTRK2 = 0.0344, ABCA4 = 0.00744, PRPH = 0, and WT1 = 0. All these genes show insufficient exon-spanning read coverage in our RNA-seq data to allow splicing analysis. We successfully detected all genes tested except PRPH and WT1. Aberrant splicing was detected in SYN1, TCF4, NTRK2, TTN, and TERT VUSs. Therefore, our results show short amplicon RT-PCR is a useful alternative for the analysis of splicing events in genes with low TPM in blood RNA for clinical diagnostics.

• Klíčová slova
• RNA-seq, RT-PCR, VUS, aberrant splicing, blood RNA,
• MeSH
• ABC transportéry genetika   MeSH
• alternativní sestřih * MeSH
• lidé MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• reverzní transkripce MeSH
• RNA * genetika   MeSH
• sestřih RNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ABC transportéry MeSH
• ABCA4 protein, human MeSH Prohlížeč
• RNA * MeSH

Giardia lambliacauses giardiasis, one of the most common human infectious diseases globally. Previous studies from our lab have shown that hsp90 gene ofGiardia is split into two halves, namely hspN and hspC. The independent pre-mRNAs of these split genes join by trans-splicing, producing a full-length Hsp90 (FlHsp90) mRNA. Genetic manipulation of the participating genes is necessary to understand the mechanism and significance of such trans-splicing based expression of Hsp90. In this study, we have performed transfection based exogenous expression of hspN and/or hspC in G. lamblia. We electroporated a plasmid containing the Avi-tagged hspN component of Hsp90 and examined its fate in G. lamblia. We show that the exogenously expressed hspN RNA gets trans-spliced to endogenously expressed hspC RNA, giving rise to a hybrid-FlHsp90. We highlight the importance of cis-elements in this trans-splicing reaction through mutational analysis. The episomal plasmid carrying deletions in the intronic region of hspN, showed inhibition of the trans-splicing reaction.Additionally, exogenous hspC RNA also followed the same fate as of exogenous hspN, while upon co-transfection with episomal hspN, they underwent trans-splicing with each other. Using eGFP as a test protein, we have shown that intronic sequences of hsp90 gene can guide trans-splicing mediated repair of any associated exonic sequences. Our study provides in vivo validation of Hsp90 trans-splicing, showing crucial role of cis-elements and importantly highlights the potential of hsp90 intronic sequences to function as a minimal splicing tool.

• Klíčová slova
• Gene expression, Giardia lamblia, Hsp90, RNA splicing, Transfection,
• MeSH
• Giardia lamblia * genetika   MeSH
• introny genetika   MeSH
• prekurzory RNA genetika   MeSH
• proteiny tepelného šoku HSP90 * genetika   MeSH
• protozoální proteiny * genetika   MeSH
• trans-splicing * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• prekurzory RNA MeSH
• proteiny tepelného šoku HSP90 * MeSH
• protozoální proteiny * MeSH