scholarly journals Yeast Ssd1 is a non-enzymatic member of the RNase II family with an alternative RNA recognition interface

2020 ◽  
Author(s):  
Rosemary A. Bayne ◽  
Uma Jayachandran ◽  
Aleksandra Kasprowicz ◽  
Stefan Bresson ◽  
David Tollervey ◽  
...  
Keyword(s):  
Stress Responses ◽  
Rna Binding ◽  
Regulatory Elements ◽  
Binding Activity ◽  
Regulatory Sequences ◽  
Rna Recognition ◽  
Rnase Ii ◽  
Sequence Requirements

AbstractThe conserved fungal RNA binding protein Ssd1, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and may act by suppressing translation of associated mRNAs. Previous studies identified motifs that are enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not well understood. Here we present the crystal structure of Ssd1 at 1.9 Å resolution. Active RNase II enzymes have a characteristic, internal RNA binding path, but in Ssd1 this is blocked by remnants of regulatory sequences. Instead, RNA binding activity has likely been relocated to the outer surface of the protein. Using in vivo crosslinking and cDNA analysis (CRAC), we identify Ssd1-RNA binding sites. These are strongly enriched in 5’UTRs of a subset of mRNAs encoding cell wall proteins. Based on these and previous analyses, we identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. These studies provide a new framework for understanding the function of a pleiotropic post-transcriptional regulator of gene expression and give insights into the evolution of regulatory elements in the RNase II family.

scholarly journals CUS2, a Yeast Homolog of Human Tat-SF1, Rescues Function of Misfolded U2 through an Unusual RNA Recognition Motif

1998 ◽  
Vol 18 (9) ◽  
pp. 5000-5009 ◽  
Author(s):  
Dong Yan ◽  
Rhonda Perriman ◽  
Haller Igel ◽  
Kenneth J. Howe ◽  
Megan Neville ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Normal Activity ◽  
Binding Activity ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Recognition Motif ◽  
Yeast Homolog

ABSTRACT A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins. CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a. Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly. Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2. Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2. Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

scholarly journals The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo

2021 ◽  
Author(s):  
Giuliano Crispatzu ◽  
Rizwan Rehimi ◽  
Tomas Pachano ◽  
Tore Bleckwehl ◽  
Sara de la Cruz Molina ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Regulatory Elements ◽  
Small Subset ◽  
Regulatory Sequences ◽  
Pluripotent Cells ◽  
Developmental Genes ◽  
Topological Features ◽  
Regulatory Properties

AbstractPoised enhancers (PEs) represent a limited and genetically distinct set of distal regulatory elements that control the induction of developmental genes in a hierarchical and non-redundant manner. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally identified and subsequently characterized using embryonic stem cells (ESC) as an in vitro differentiation system, it is currently unknown whether PEs are functionally conserved in vivo. Here, we generate and mine various types of genomic data to show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that, in mouse pluripotent cells, the interactions between PEs and their bivalent target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. proximity to CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation, preferentially within specific vertebrate clades, with only a small subset being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting evolutionary conserved PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential and non-redundant roles during the induction of major developmental genes in vivo.

RNA binding by Sxl proteins in vitro and in vivo

1994 ◽  
Vol 14 (7) ◽  
pp. 4975-4990
Author(s):  
M E Samuels ◽  
D Bopp ◽  
R A Colvin ◽  
R F Roscigno ◽  
M A Garcia-Blanco ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Rna Binding ◽  
Polytene Chromosomes ◽  
Regulatory Sequences ◽  
Acceptor Site ◽  
Nuclear Extracts ◽  
Preferential Binding

Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.

scholarly journals Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements

2016 ◽  
Author(s):  
Molly Gasperini ◽  
Gregory M. Findlay ◽  
Aaron McKenna ◽  
Jennifer H. Milbank ◽  
Choli Lee ◽  
...  
Keyword(s):  
Large Scale ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Mendelian Disease ◽  
Regulatory Sequences ◽  
Coding Region ◽  
Endogenous Gene ◽  
Distal Regulatory Elements

AbstractThe extent to which distal non-coding mutations contribute to Mendelian disease remains a major unknown in human genetics. Given that a gene’s in vivo function can be appropriately modeled in vitro, CRISPR/Cas9 genome editing enables the large-scale perturbation of distal non-coding regions to identify functional elements in their native context. However, early attempts at such screens have relied on one individual guide RNA (gRNA) per cell, resulting in sparse mutagenesis with minimal redundancy across regions of interest. To address this, we developed a system that uses pairs of gRNAs to program thousands of kilobase-scale deletions that scan across a targeted region in a tiling fashion (“ScanDel”). As a proof-of-concept, we applied ScanDel to program 4,342 overlapping 1- and 2- kilobase (Kb) deletions that tile a 206 Kb region centered on HPRT1, the gene underlying Lesch-Nyhan syndrome, with median 27-fold redundancy per base. Programmed deletions were functionally assayed by selecting for loss of HPRT1 function with 6-thioguanine. HPRT1 exons served as positive controls, and all were successfully identified as functionally critical by the screen. Remarkably, HPRT1 function appeared robust to deletion of any intergenic or deeply intronic non-coding region across the 206 Kb locus, indicating that proximal regulatory sequences are sufficient for its expression. A sparser mutagenesis screen of the same 206 Kb with individual gRNAs also failed to identify critical distal regulatory elements. Although our screen did find programmed deletions and individual gRNAs with putative functional consequences that targeted exon-proximal non-coding sequences (e.g. the promoter), long-read sequencing revealed that this signal was driven almost entirely by rare, unexpected deletions that extended into exonic sequence. These targeted validation experiments defined a small region surrounding the transcriptional start site as the only non-coding sequence essential to HPRT1 function. Overall, our results suggest that distal regulatory elements are not critical for HPRT1 expression, and underscore the necessity of comprehensive edited-locus genotyping for validating the results of CRISPR screens. The application of ScanDel to additional loci will enable more insight into the extent to which the disruption of distal non-coding elements contributes to Mendelian diseases. In addition, dense, redundant, large-scale deletion scanning with gRNA pairs will facilitate a deeper understanding of endogenous gene regulation in the human genome.

scholarly journals Two Pax-binding sites are required for early embryonic brain expression of an Engrailed-2 transgene

Development ◽  
1996 ◽  
Vol 122 (2) ◽  
pp. 627-635 ◽  
Author(s):  
D.L. Song ◽  
G. Chalepakis ◽  
P. Gruss ◽  
A.L. Joyner
Keyword(s):  
Dna Binding ◽  
Transgenic Mice ◽  
Binding Sites ◽  
Regulatory Elements ◽  
Developing Brain ◽  
Molecular Evidence ◽  
Regulatory Sequences ◽  
Pax Genes

The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. The regulation of En-2 expression was studied using in vitro protein-DNA binding assays and in vivo expression analysis in transgenic mice to gain insight into the genetic events that lead to regionalization of the developing brain. A minimum En-2 1.0 kb enhancer fragment was defined and found to contain multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. Furthermore, the mid-hindbrain regulatory sequences were shown to be structurally and functionally conserved in humans. The mouse paired-box-containing genes Pax-2, Pax-5 and Pax-8 show overlapping expression with the En genes in the developing brain. Significantly, two DNA-binding sites for Pax-2, Pax-5 and Pax-8 proteins were identified in the 1.0 kb En-2 regulatory sequences, and mutation of the binding sites disrupted initiation and maintenance of expression in transgenic mice. These results present strong molecular evidence that the Pax genes are direct upstream regulators of En-2 in the genetic cascade controlling mid-hindbrain development. These mouse studies, taken together with others in Drosophila and zebrafish on the role of Pax genes in controlling expression of En family members, indicate that a Pax-En genetic pathway has been conserved during evolution.

The mitotic phosphorylation of p54nrb modulates its RNA binding activity

2011 ◽  
Vol 89 (4) ◽  
pp. 423-433 ◽  
Author(s):  
Céline Bruelle ◽  
Mikaël Bédard ◽  
Stéphanie Blier ◽  
Martin Gauthier ◽  
Abdulmaged M. Traish ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Binding Assays ◽  
Non Coding Rna ◽  
Protein Partners ◽  
Biochemical Assays ◽  
Mitotic Phosphorylation

The RNA-binding protein p54nrb is involved in many nuclear processes including transcription, RNA processing, and retention of hyperedited RNAs. In interphase cells, p54nrb localizes to the nucleoplasm and concentrates with protein partners in the paraspeckles via an interaction with the non-coding RNA Neat1. During mitosis, p54nrb becomes multiphosphorylated and the effects of this modification are not known. In the present study, we show that p54nrb phosphorylation does not affect the interactions with its protein partners but rather diminishes its general RNA-binding ability. Biochemical assays indicate that in vitro phosphorylation of a GST-p54nrb construct by CDK1 abolishes the interaction with 5′ splice site RNA sequence. Site-directed mutagenesis shows that the threonine 15 residue, located N-terminal to the RRM tandem domains of p54nrb, is involved in this inhibition. In vivo analysis reveals that Neat1 ncRNA co-immunoprecipitates with p54nrb in either interphase or mitotic cells, suggesting that p54nrb–Neat1 interaction is not modulated by phosphorylation. Accordingly, in vitro phosphorylated GST-p54nrb still interacts with PIR-1 RNA, a G-rich Neat1 sequence known to interact with p54nrb. In vitro RNA binding assays show that CDK1-phosphorylation of a GST-p54nrb construct abolishes its interaction with homoribopolymers poly(A), poly(C), and poly(U) but not with poly(G). These data suggest that p54nrb interaction with RNA could be selectively modulated by phosphorylation during mitosis.

scholarly journals The 5' leader of a chloroplast mRNA mediates the translational requirements for two nucleus-encoded functions in Chlamydomonas reinhardtii.

1994 ◽  
Vol 14 (8) ◽  
pp. 5268-5277 ◽  
Author(s):  
W Zerges ◽  
J D Rochaix
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Rna Binding ◽  
Mrna Translation ◽  
Binding Activity ◽  
Leader Sequence ◽  
Sequence Motif ◽  
Wild Type ◽  
Chloroplast Mrna

In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previously shown to abolish the synthesis of the photosystem II core polypeptide subunit P6, which is encoded by the chloroplast psbC gene. In this report the functions encoded by F34 and F64 are shown to be required for translation of the psbC mRNA, on the basis of the finding that the expression of a heterologous reporter gene fused to the psbC 5' nontranslated leader sequence requires wild-type F34 and F64 alleles in vivo. Moreover, a point mutation in the psbC 5' nontranslated leader sequence suppresses this requirement for wild-type F34 function. In vitro RNA-protein cross-linking studies reveal that chloroplast protein extracts from strains carrying the F64 mutation contain an approximately 46-kDa RNA-binding protein. The absence of the RNA-binding activity of this protein in chloroplast extracts of wild-type strains suggests that it is related to the role of the F64-encoded function for psbC mRNA translation. The binding specificity of this protein appears to be for an AU-rich RNA sequence motif.

scholarly journals RNA binding by Sxl proteins in vitro and in vivo.

1994 ◽  
Vol 14 (7) ◽  
pp. 4975-4990 ◽  
Author(s):  
M E Samuels ◽  
D Bopp ◽  
R A Colvin ◽  
R F Roscigno ◽  
M A Garcia-Blanco ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Rna Binding ◽  
Polytene Chromosomes ◽  
Regulatory Sequences ◽  
Acceptor Site ◽  
Nuclear Extracts ◽  
Preferential Binding

Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.

ARCD-1, an apobec-1-related cytidine deaminase, exerts a dominant negative effect on C to U RNA editing

2001 ◽  
Vol 281 (6) ◽  
pp. C1904-C1916 ◽  
Author(s):  
Shrikant Anant ◽  
Debnath Mukhopadhyay ◽  
Vakadappu Sankaranand ◽  
Susan Kennedy ◽  
Jeffrey O. Henderson ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Cytidine Deaminase ◽  
Binding Activity ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Mrna Editing ◽  
Apob Mrna

Mammalian apolipoprotein B (apoB) C to U RNA editing is catalyzed by a multicomponent holoenzyme containing a single catalytic subunit, apobec-1. We have characterized an apobec-1 homologue, ARCD-1, located on chromosome 6p21.1, and determined its role in apoB mRNA editing. ARCD-1 mRNA is ubiquitously expressed; phylogenetic analysis reveals it to be a distant member of the RNA editing family. Recombinant ARCD-1 demonstrates cytidine deaminase and apoB RNA binding activity but does not catalyze C to U RNA editing, either in vitro or in vivo. Although not competent itself to mediate deamination of apoB mRNA, ARCD-1 inhibits apobec-1-mediated C to U RNA editing. ARCD-1 interacts and heterodimerizes with both apobec-1 and apobec-1 complementation factor (ACF) and localizes to both the nucleus and cytoplasm of transfected cells. Together, the data suggest that ARCD-1 is a novel cytidine deaminase that interacts with apobec-1 and ACF to inhibit apoB mRNA editing, possibly through interaction with other protein components of the apoB RNA editing holoenzyme.

Sign in / Sign up

Export Citation Format

Share Document