Učni načrt predmeta

Predmet:

Interaktomika bioloških makromolekul

Course:

Biomacromolecular interactomics

Študijski program in stopnja / Study programme and level	Študijska smer / Study field	Letnik / Academic year	Semester / Semester
Nanoznanosti in nanotehnologije, 3. stopnja		1	1
Nanosciences and nanotechnologies, 3rd cycle		1	1

Vrsta predmeta / Course type

Izbirni / Elective

Univerzitetna koda predmeta / University course code:

NANO3-935

Predavanja Lectures	Seminar Seminar	Vaje Tutorial	Klinične vaje work	Druge oblike študija	Samost. delo Individ. work	ECTS
15	15			15	105	5

*Navedena porazdelitev ur velja, če je vpisanih vsaj 15 študentov. Drugače se obseg izvedbe kontaktnih ur sorazmerno zmanjša in prenese v samostojno delo. / This distribution of hours is valid if at least 15 students are enrolled. Otherwise the contact hours are linearly reduced and transfered to individual work.

Nosilec predmeta / Course leader:

prof. dr. Boris Rogelj

Sodelavci / Lecturers:

Jeziki / Languages:

Predavanja / Lectures:

slovenščina, angleščina / Slovenian, English

Vaje / Tutorial:

Pogoji za vključitev v delo oz. za opravljanje študijskih obveznosti:

Prerequisites:

Zaključen študij druge stopnje s področja naravoslovja ali tehnologije ali zaključen študij druge stopnje na drugih področjih z znanjem osnov s področja predmeta.

Completed second cycle studies in natural sciences or technologies or completed second cycle studies in other fields with knowledge of fundamentals in the field of this course.

Vsebina:

Content (Syllabus outline):

1. Uvod v interakcije protein-protein (PPI), protein-RNA (PRI) in RNA-RNA (RRI). Biomedicinske posledice interakcij.
2. Presejalne metode za določanje interakcij proteinov in RNA (Bližinska biotinilacija, ko-imunoprecipitacija, Y2H, Y3H, afinitetna precipitacija, mikromreže, CLIP, PARIS, COMRADES, …)
3. Biokemijska validacija interakcij (co-ip, affinitetna precipitacija,…)
4. Biofizična validacija interakcij (termoforeza, BLI, SPR,…)
5. Celična validacija interakcije (kolokalizacija, PLA, bližinska biotinilacija)
6. Interaktomsko načrtovanje delovnega toka presejalnega poskusa.
7. Praktično dela na beljakovinah/RNA po izbiri z izbiro naslednjih protokolov:
a. PPI: Co-IP, proximity biotinilation (BioID, APEX, BAR), diferencialno centrifugiranje, afiniteta pull-down
b. PRI: RIP, CLIP, RNA pulldown,
c. RRI: COMRADES, PARIS
d. Validacija: co-IP, ko-lokalizacija, termoforeza, PLA,
e. drugo

1. Introduction to protein-protein (PPI), protein-RNA (PRI) and RNA-RNA (RRI) interactions. Biomedical implications of the interactions.
2. Screening methods to determine protein and RNA interactions (Proximity biotinylation, co-immunoprecipitation, Y2H, Y3H, affinity pulldowns, microarrays, CLIP, RBPome PARIS, COMRADES, …)
3. Biochemical validation of interactions. (co-IP, affinity pulldown,…)
4. Biophysical validation of the interactions (thermophoresis, BLI, SPR,…)
5. Cellular validation of interaction (kolokalizacija, PLA, bližinska biotinilacija)
6. Interactomic screening workflow design.
7. Practical work on protein/RNA of choice using a selection of the following protocols:
a. PPI: Co-IP, proximity biotinylation (BioID, APEX, BAR), differential centrifugation, affinity pull-down
b. PRI: RIP, CLIP, RNA pulldown,
c. RRI: COMRADES, PARIS
d. Validation: co-IP, co-localization, thermophoresis, PLA,
e. other

Temeljna literatura in viri / Readings:

1. Proximity-dependent labeling methods for proteomic profiling in living cells: An update. Bosch JA, Chen CL, Perrimon N. Wiley Interdiscip Rev Dev Biol. 2021 Jan;10(1):e392. doi: 10.1002/wdev.392. Epub 2020 Sep 10. PMID: 32909689
2. Technologies for the identification and validation of protein-protein interactions. Pichlerova K, Hanes J. Gen Physiol Biophys. 2021 Nov;40(6):495-522. doi: 10.4149/gpb_2021035. PMID: 34897023
3. RNA-Centric Methods: Toward the Interactome of Specific RNA Transcripts. Gräwe C, Stelloo S, van Hout FAH, Vermeulen M. Trends Biotechnol. 2021 Sep;39(9):890-900. doi: 10.1016/j.tibtech.2020.11.011. Epub 2020 Dec 19. PMID: 33353763
4. Compendium of Methods to Uncover RNA-Protein Interactions In Vivo. Majumder M, Palanisamy V. Methods Protoc. 2021 Mar 19;4(1):22. doi: 10.3390/mps4010022. PMID: 33808611
5. RNA interactomics: recent advances and remaining challenges. Schönberger B, Schaal C, Schäfer R, Voß B. F1000Res. 2018 Nov 20;7:F1000 Faculty Rev-1824. doi: 10.12688/f1000research.16146.1. eCollection 2018. PMID: 30519453

Cilji in kompetence:

Objectives and competences:

Cilj predmeta je spoznati napredne eksperimentalne tehnike določanja ter karakterizacija proteinskih in RNA interakcij in uporabnost v biomedicinskih raziskavah.
Pridobljene bodo praktične izkušnje z izbranim potekom dela in metodologijami, ki se bodo izvajala. Po končanem tečaju bi morali biti študenti sposobni izbrati najprimernejše presejalne tehnike in validacije, oblikovati takšne eksperimente ter pridobiti globlji vpogled in izkušnje v poteku dela, povezanem s praktičnim delom predmeta.

The course will familiarize graduate students with advanced experimental techniques for determination and characterization of protein and RNA interactions and their utility in biomedical research. Practical experience will be gained on selected workflow and methodologies that will be carried out.
After completing the course, students should be able to select the most appropriate screening and validation techniques, design such experiments and gain deeper insight and experience in the workflow associated with the practical part of the course.

Predvideni študijski rezultati:

Intendeded learning outcomes:

Znanje in razumevanje: Poznavanje razpoložljivih osnovnih in naprednih interaktomskih orodij in eksperimentov. Oblikovanje interaktomskih eksperimentalnih delovnih tokov. Izkušnje v izbranem interaktomskem delovnem toku.

Uporaba: Tečaj bo izveden kot kratka serija predavanj, ki ji bo sledilo načrtovanje in eksperimentalno delo na interaktomskem delovnem toku po izbiri.

Refleksija: Razumevanje osnov interaktomike.

Prenosljive spretnosti: Razumevanje in uporaba izbranih interaktomskih metodologij.

Knowledge and understanding: Familiarity with available basic and advanced interactomic tools and experiments. Design of interactomic experimental workflows. Experience in a selected interactomic workflow.

Application: The course will be carried out as a short lecture serries followed by design and experimental work on interactomic workflow of choice.

Reflection: Understanding of basics of interactomics.

Transferable skills: Understanding and use of selected interactomic methodologies.

Metode poučevanja in učenja:

Learning and teaching methods:

Predavanja, seminar, konzultacije, individualno delo

Lectures, seminar work, consultations, individual work

Načini ocenjevanja:

Delež v % / Weight in %

Assesment:

Seminar

50 %

Seminar

Ustni zagovor seminarja

50 %

Oral defense of seminar work

Reference nosilca / Lecturer's references:

1.	MALNAR, Mirjana, …, ROGELJ, Boris. Phenylalanine-tRNA aminoacylation is compromised by ALS/FTD-associated C9orf72 C4G2 repeat RNA. Nature communications. 2023, DOI: 10.1038/s41467-023-41511-3. [COBISS.SI-ID 166968323]
2.	MOTALN, Helena, …, ROGELJ, Boris. Abl kinase-mediated FUS Tyr526 phosphorylation alters nucleocytoplasmic FUS localization in FTLD-FUS. Brain : journal of neurology. 2023, DOI: 10.1093/brain/awad130. [COBISS.SI-ID 151014403]
3.	BOŽIČ, Janja, …, ROGELJ, Boris. Interactome screening of C9orf72 dipeptide repeats reveals VCP sequestration and functional impairment by polyGA. Brain : journal of neurology. 2022, DOI: 10.1093/brain/awab300. [COBISS.SI-ID 76933379]
4.	MALNAR, Mirjana & ROGELJ, Boris. SFPQ regulates the accumulation of RNA foci and dipeptide repeat proteins from the expanded repeat mutation in C9orf72. Journal of cell science. 2021, DOI: 10.1242/jcs.256602. [COBISS.SI-ID 50363907]
5.	BAJC ČESNIK, Ana, …, ROGELJ, Boris. Nuclear RNA foci from C9ORF72 expansion mutation form paraspeckle-like bodies. Journal of cell science. 2019, DOI: 10.1242/jcs.224303. [COBISS.SI-ID 32174887]