Choudhary, Bikash et al. (2021) International Worm Meeting "Spliceosomal component PRP-40 regulates alternative splicing of microexons."

Norris, Adam, Marx, Olivia, Choudhary, Bikash

[International Worm Meeting, 2021]

Alternative splicing plays an important role in cellular diversity and is executed by a specialized splicing machinery, the spliceosome. Splicing of very short "microexons" (less_than_or_equal 51 nucleotides) is a mechanistic challenge for the spliceosome due to their short length. Therefore, our understanding of microexon splicing has remained poor. By performing a forward genetic screen for regulators of UNC-16/JIP3 alternative splicing, we have identified novel regulators of cassette exon splicing. One candidate, prp-40 (Pre-mRNA Processing Factor), encodes an essential component of the U1 snRNP of the spliceosomal complex. We show that an interaction between prp-40 and exc-7/ELAV, a RNA binding protein (RBP)-encoding gene, governs the splicing of the cassette exon. In addition, through transcriptomic analysis of prp-40 loss-of-function mutants, we determined that prp-40 affects alternative splicing, but not constitutive spicing. Loss of prp-40 had the strongest effect on microexons. In the absence of prp-40, nearly all microexons were reduced to undetectable levels, indicating that prp-40 is a central regulator of microexon splicing. Furthermore, gene ontology analysis of genes with altered splicing profiles showed an enrichment of components of the neuronal synaptic signaling machinery, indicating a potential role for prp-40 in neurological disorders. Subsequent analyses in pursuit of the underlying mechanism revealed that prp-40-mediated microexon splicing is influenced by the length of introns flanking the microexon and by the length of the microexon itself. Shortening of the upstream intron or lengthening of the microexon leads to a prp-40-independent inclusion of the microexons, establishing that prp-40 regulates microexon splicing in a length-dependent manner. Finally, in order to understand if the function of prp-40 is evolutionarily conserved, we performed a knockdown of PRPF40A in a mouse neuroblastoma cell line. Indeed, we found that the splicing of a set of microexons was perturbed, whereas that of normal-sized exons remained largely unaffected. Thus, prp-40 performs an evolutionarily conserved function in alternative splicing.

Choudhary, Bikash C et al. (2011) International Worm Meeting "JIP3/UNC-16 has an UNC-101 dependent role in synaptic vesicle biogenesis."

Fukuzono, Takashi, Choudhary, Bikash C, Hisamoto, Naoki, Matsumoto, Kunihiro, Koushika, Sandhya P, Kumar, Jitendra, Chun, Li

[International Worm Meeting, 2011]

UNC-16/JIP3 is a known cargo adapter for the Kinesin-I (UNC-116) motor and a Jun Kinase (JNK) scaffolding protein. unc-16/JIP3 mutants are able to bypass the requirement for the UNC-104/KIF1A kinesin motor for transport of pre-synaptic vesicle protein. In addtion, in unc-16 animals we observe (i) Synaptobrevin and RAB-3 moving in aberrant large tubular compartments that emerge from the cell body, (ii) Synaptobrevin is present at the dendritic tips of chemosensory neurons and (iii) resident golgi enzymes are mis-localized and present along the entire neuronal process. These data suggest a role for UNC-16 in trafficking likely at the golgi. The mis-localization of Synaptobrevin to dendrites of chemosensory neurons and large moving tubular compartments containing RAB-3 are also present in lrk-1 mutants. However in lrk-1 animals localization of resident golgi enzymes is not greatly altered and lrk-1 is unable to bypass the requirement of UNC-104 in the localization of synaptic vesicle proteins. The dendritic localization of Synaptobrevin in lrk-1 has been shown to be unc-101 dependent. However, the dendritic localization of Synaptobrevin in unc-16 animals is unc-101 dependent only in an unc-16 allele with a late stop in its ORF. Our data suggest a novel role for UNC-16 in the cell body where it acts either upstream or in parallel with LRK-1 in an UNC-101 dependent pathway in formation of the synaptic vesicle protein transport carriers.

Choudhary, Bikash et al. (2015) International Worm Meeting "UNC-16 regulates early steps of synaptic vesicle protein trafficking via LRK-1 and UNC-101."

Kamak, Madhushree, Chun, Li, Matsumoto, Kunihiro, Nguyen, Ken, Awasthi, Anjali, Fukuzono, Takashi, Koushika, Sandhya, Hisamoto, Naoki, Kumar, Jitendra, Choudhary, Bikash

[International Worm Meeting, 2015]

Synaptic vesicle protein trafficking occurs via vesicular cargo compartments formed at the cell body of the neuron. The identity of the cargo compartment as a precursor of a synaptic vesicle is determined by multiple factors. It carries a defined set of synaptic vesicle proteins such as RAB-3 and SNB-1,is transported by molecular motors such as UNC-104 and dynein in the axon. Our study has identified UNC-16 as a regulator of early steps in synaptic vesicle protein trafficking. In the absence of UNC-16,we observe that vesicular cargo exit the cell body into the neuronal process, carrying both synaptic vesicle proteins and Golgi resident enzymes.These aberrant vesicles are larger than wild type cargo compartments and atypically enter the dendritic process. We propose that the altered nature of the cargo compartment-both content and size- leads to the altered motor selectivity and mistrafficking phenotypes previously observed by Byrd et al (2001).This study demonstrates that the formation of aberrant vesicular cargo compartments in unc-16 is dependent on the function of LRK-1- a homolog of the familial Parkinsonism gene LRRK2. lrk-1 mutants exhibit similar atypical cargo compartments exiting the neuronal cell body.The over-expression of LRK-1 in unc-16 is able to rescue a subset of the trafficking defects. LRK-1 inhibits the trafficking of synaptic vesicles into the dendrite via UNC-101- an AP-1 mu1 clathrin adaptor (Sakaguchi-Nakashima et al, 2007). We show that the localisation of UNC-101 on the Golgi is altered in both unc-16 and lrk-1 animals.This mislocalisation can be rescued in unc-16 by the over-expression of LRK-1. This suggests that the formation of an appropriate synaptic vesicle protein cargo compartment depends on the golgi localisation of UNC-101 regulated by UNC-16 at least partially through LRK-1.Interpreting a set of genetic interactions and biochemical evidence; we propose a model in which UNC-16 and LRK-1 present at the Golgi regulate transport carrier biogenesis. They maintain the identity and thereby motor selectivity of the synaptic vesicle protein trafficking compartment.