In eukaryotic cells, the genomic deoxyribonucleic acid (DNA) in the nucleus is separated from the... more In eukaryotic cells, the genomic deoxyribonucleic acid (DNA) in the nucleus is separated from the translational machinery in the cytoplasm by the nuclear envelope. Transport of macromolecules such as proteins and ribonucleic acid (RNA) across this membrane is essential for cellular function and requires active nuclear–cytoplasmic transport systems. These systems consist of soluble transport receptors, which recognize and bind cargo in one compartment, mediate transport through nuclear pore complexes embedded in the nuclear envelope and deliver cargo in the target compartment. Disruption of this highly regulated process results in abnormal cell function and is linked to human disease etiology. Understanding the contribution of nuclear protein and RNA transport to cellular organization is one of the major challenges in cell biology. Key Concepts Transport into and out of the nucleus occurs through nuclear pore complexes (NPCs), which are large proteinaceous channels that perforate the nuclear membrane. Proteins destined for import into the nucleus contain a nuclear localization signal (NLS) and proteins destined for export from the nucleus contain a nuclear export signal (NES), each of which targets them for transport. Soluble transport receptors called importins and exportins or karyopherins recognize and bind macromolecular cargos and facilitate transport through the nuclear pore. The asymmetric distribution of RanGDP in the cytoplasm and RanGTP in the nucleus controls the directionality of nuclear transport. Nuclear protein import can be regulated through inter- or intramolecular occlusion of the NLS or NES; posttranslation modification of the targeting signal; compartmental sequestration of the cargo protein or altering properties of the nuclear transport machinery including receptors and nuclear pores. Many classes of RNA are transported via Ran-regulated, karyopherin-dependent pathways. Messenger ribonucleic acid (mRNA) export is highly coupled to mRNA processing and is mediated by distinct receptors. Mutations in nuclear targeting signals and nuclear transport receptors have been linked to several human diseases. Keywords: nuclear transport; importin; karyopherin; nuclear pore complex; nucleocytoplasmic trafficking
In eukaryotic cells, RNA synthesis in the nucleus is separated from protein synthesis in the cyto... more In eukaryotic cells, RNA synthesis in the nucleus is separated from protein synthesis in the cytoplasm by the nuclear envelope. Against a background of emerging information concerning the molecular mechanisms of nucleo-cytoplasmic transport, understanding the contribution of this exchange to cellular organization is one of the major challenges in cell biology. Keywords: protein trafficking; RNA transport; nuclear transport; nuclear pore; nucleus
TheDrosophilapolyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost... more TheDrosophilapolyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls axon projection, locomotion, and memory. Here we define an unexpectedly specific role for Nab2 in regulating splicing of ∼150 exons/introns in the head transcriptome and link the most prominent of these, female retention of a male-specific exon in the sex determination factorSex-lethal(Sxl), to a role in m6A-dependent mRNA splicing. Genetic evidence indicates that aberrantSxlsplicing underlies multiple phenotypes inNab2mutant females. At a molecular level, Nab2 associates withSxlpre-mRNA and ensures proper female-specific splicing by preventing m6A hypermethylation by Mettl3 methyltransferase. Consistent with these results, reducing Mettl3 expression rescues developmental, behavioral and neuroanatomical phenotypes inNab2mutants. Overall these data identify Nab2 as a required regulator of m6A-regulatedSxlsplicing and imply a broade...
Skeletal muscle is primarily composed of large myofibers containing thousands of post-mitotic nuc... more Skeletal muscle is primarily composed of large myofibers containing thousands of post-mitotic nuclei distributed throughout a common cytoplasm. Protein production and localization in specialized myofiber regions is crucial for muscle function. Myonuclei differ in transcriptional activity and protein accumulation, but how these differences among nuclei sharing a cytoplasm are achieved is unknown. Regulated nuclear import of proteins is one potential mechanism for regulating transcription spatially and temporally in individual myonuclei. The best-characterized nuclear localization signal (NLS) in proteins is the classical NLS (cNLS), but many other NLS motifs exist. We examined cNLS and non-cNLS reporter protein import using multinucleated muscle cells generated , revealing that cNLS and non-cNLS nuclear import differs among nuclei in the same cell. Investigation of cNLS nuclear import rates in isolated myofibers confirmed differences in nuclear import rates among myonuclei. Analyzing...
The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein requir... more The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. Here, we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-s...
DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-s... more DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and exten...
A well-written application for funding in support of basic biological or biomedical research or i... more A well-written application for funding in support of basic biological or biomedical research or individual training fellowship requires that the author perform several functions well. They must (1) identify an important topic, (2) provide a brief but persuasive introduction to highlight its significance, (3) identify one or two key questions that if answered would impact the field, (4) present a series of logical experiments and convince the reader that the approaches are feasible, doable within a certain period of time, and have the potential to answer the questions posed, and (5) include citations that demonstrate both scholarship and an appropriate command of the relevant literature and techniques involved in the proposed research study. In addition, preparation of any compelling application requires formal scientific writing and editing skills that are invaluable in any career. These are also all key components in a doctoral dissertation and encompass many of the skills that we ...
This chapter discusses base excision repair (BER) and the known mechanisms defined thus far regul... more This chapter discusses base excision repair (BER) and the known mechanisms defined thus far regulating BER in eukaryotes. Unlike the situation with nucleotide excision repair and double-strand break repair, little is known about how BER is regulated to allow for efficient and accurate repair of many types of DNA base damage in both nuclear and mitochondrial genomes. Regulation of BER has been proposed to occur at multiple, different levels including transcription, posttranslational modification, protein-protein interactions, and protein localization; however, none of these regulatory mechanisms characterized thus far affect a large spectrum of BER proteins. This chapter discusses a recently discovered mode of BER regulation defined in budding yeast cells that involves mobilization of DNA repair proteins to DNA-containing organelles in response to genotoxic stress.
BackgroundThe nuclear poly(A) binding protein 1 (PABPN1) is a ubiquitously expressed proteinthat ... more BackgroundThe nuclear poly(A) binding protein 1 (PABPN1) is a ubiquitously expressed proteinthat plays critical roles at multiple steps in post-transcriptional regulation ofgene expression. Short expansions of the polyalanine tract in the N-terminus ofPABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onsetdisease characterized by eyelid drooping, difficulty in swallowing, and weaknessin the proximal limb muscles. Why alanine-expanded PABPN1 leads to muscle-specificpathology is unknown. Given the general function of PABPN1 in RNA metabolism,intrinsic characteristics of skeletal muscle may make this tissue susceptible tothe effects of mutant PABPN1.MethodsTo begin to understand the muscle specificity of OPMD, we investigated thesteady-state levels of PABPN1 in different tissues of humans and mice.Additionally, we analyzed the levels of PABPN1 during muscle regeneration afterinjury in mice. Furthermore, we assessed the dynamics of PABPN1 mRNA decay inskeletal ...
In eukaryotic cells, the genomic deoxyribonucleic acid (DNA) in the nucleus is separated from the... more In eukaryotic cells, the genomic deoxyribonucleic acid (DNA) in the nucleus is separated from the translational machinery in the cytoplasm by the nuclear envelope. Transport of macromolecules such as proteins and ribonucleic acid (RNA) across this membrane is essential for cellular function and requires active nuclear–cytoplasmic transport systems. These systems consist of soluble transport receptors, which recognize and bind cargo in one compartment, mediate transport through nuclear pore complexes embedded in the nuclear envelope and deliver cargo in the target compartment. Disruption of this highly regulated process results in abnormal cell function and is linked to human disease etiology. Understanding the contribution of nuclear protein and RNA transport to cellular organization is one of the major challenges in cell biology. Key Concepts Transport into and out of the nucleus occurs through nuclear pore complexes (NPCs), which are large proteinaceous channels that perforate the nuclear membrane. Proteins destined for import into the nucleus contain a nuclear localization signal (NLS) and proteins destined for export from the nucleus contain a nuclear export signal (NES), each of which targets them for transport. Soluble transport receptors called importins and exportins or karyopherins recognize and bind macromolecular cargos and facilitate transport through the nuclear pore. The asymmetric distribution of RanGDP in the cytoplasm and RanGTP in the nucleus controls the directionality of nuclear transport. Nuclear protein import can be regulated through inter- or intramolecular occlusion of the NLS or NES; posttranslation modification of the targeting signal; compartmental sequestration of the cargo protein or altering properties of the nuclear transport machinery including receptors and nuclear pores. Many classes of RNA are transported via Ran-regulated, karyopherin-dependent pathways. Messenger ribonucleic acid (mRNA) export is highly coupled to mRNA processing and is mediated by distinct receptors. Mutations in nuclear targeting signals and nuclear transport receptors have been linked to several human diseases. Keywords: nuclear transport; importin; karyopherin; nuclear pore complex; nucleocytoplasmic trafficking
In eukaryotic cells, RNA synthesis in the nucleus is separated from protein synthesis in the cyto... more In eukaryotic cells, RNA synthesis in the nucleus is separated from protein synthesis in the cytoplasm by the nuclear envelope. Against a background of emerging information concerning the molecular mechanisms of nucleo-cytoplasmic transport, understanding the contribution of this exchange to cellular organization is one of the major challenges in cell biology. Keywords: protein trafficking; RNA transport; nuclear transport; nuclear pore; nucleus
TheDrosophilapolyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost... more TheDrosophilapolyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls axon projection, locomotion, and memory. Here we define an unexpectedly specific role for Nab2 in regulating splicing of ∼150 exons/introns in the head transcriptome and link the most prominent of these, female retention of a male-specific exon in the sex determination factorSex-lethal(Sxl), to a role in m6A-dependent mRNA splicing. Genetic evidence indicates that aberrantSxlsplicing underlies multiple phenotypes inNab2mutant females. At a molecular level, Nab2 associates withSxlpre-mRNA and ensures proper female-specific splicing by preventing m6A hypermethylation by Mettl3 methyltransferase. Consistent with these results, reducing Mettl3 expression rescues developmental, behavioral and neuroanatomical phenotypes inNab2mutants. Overall these data identify Nab2 as a required regulator of m6A-regulatedSxlsplicing and imply a broade...
Skeletal muscle is primarily composed of large myofibers containing thousands of post-mitotic nuc... more Skeletal muscle is primarily composed of large myofibers containing thousands of post-mitotic nuclei distributed throughout a common cytoplasm. Protein production and localization in specialized myofiber regions is crucial for muscle function. Myonuclei differ in transcriptional activity and protein accumulation, but how these differences among nuclei sharing a cytoplasm are achieved is unknown. Regulated nuclear import of proteins is one potential mechanism for regulating transcription spatially and temporally in individual myonuclei. The best-characterized nuclear localization signal (NLS) in proteins is the classical NLS (cNLS), but many other NLS motifs exist. We examined cNLS and non-cNLS reporter protein import using multinucleated muscle cells generated , revealing that cNLS and non-cNLS nuclear import differs among nuclei in the same cell. Investigation of cNLS nuclear import rates in isolated myofibers confirmed differences in nuclear import rates among myonuclei. Analyzing...
The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein requir... more The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. Here, we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-s...
DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-s... more DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and exten...
A well-written application for funding in support of basic biological or biomedical research or i... more A well-written application for funding in support of basic biological or biomedical research or individual training fellowship requires that the author perform several functions well. They must (1) identify an important topic, (2) provide a brief but persuasive introduction to highlight its significance, (3) identify one or two key questions that if answered would impact the field, (4) present a series of logical experiments and convince the reader that the approaches are feasible, doable within a certain period of time, and have the potential to answer the questions posed, and (5) include citations that demonstrate both scholarship and an appropriate command of the relevant literature and techniques involved in the proposed research study. In addition, preparation of any compelling application requires formal scientific writing and editing skills that are invaluable in any career. These are also all key components in a doctoral dissertation and encompass many of the skills that we ...
This chapter discusses base excision repair (BER) and the known mechanisms defined thus far regul... more This chapter discusses base excision repair (BER) and the known mechanisms defined thus far regulating BER in eukaryotes. Unlike the situation with nucleotide excision repair and double-strand break repair, little is known about how BER is regulated to allow for efficient and accurate repair of many types of DNA base damage in both nuclear and mitochondrial genomes. Regulation of BER has been proposed to occur at multiple, different levels including transcription, posttranslational modification, protein-protein interactions, and protein localization; however, none of these regulatory mechanisms characterized thus far affect a large spectrum of BER proteins. This chapter discusses a recently discovered mode of BER regulation defined in budding yeast cells that involves mobilization of DNA repair proteins to DNA-containing organelles in response to genotoxic stress.
BackgroundThe nuclear poly(A) binding protein 1 (PABPN1) is a ubiquitously expressed proteinthat ... more BackgroundThe nuclear poly(A) binding protein 1 (PABPN1) is a ubiquitously expressed proteinthat plays critical roles at multiple steps in post-transcriptional regulation ofgene expression. Short expansions of the polyalanine tract in the N-terminus ofPABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onsetdisease characterized by eyelid drooping, difficulty in swallowing, and weaknessin the proximal limb muscles. Why alanine-expanded PABPN1 leads to muscle-specificpathology is unknown. Given the general function of PABPN1 in RNA metabolism,intrinsic characteristics of skeletal muscle may make this tissue susceptible tothe effects of mutant PABPN1.MethodsTo begin to understand the muscle specificity of OPMD, we investigated thesteady-state levels of PABPN1 in different tissues of humans and mice.Additionally, we analyzed the levels of PABPN1 during muscle regeneration afterinjury in mice. Furthermore, we assessed the dynamics of PABPN1 mRNA decay inskeletal ...
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